HK1128732A - Detection of nucleic acids by target-specific hybrid capture method - Google Patents

Description

Detection of nucleic acids by target-specific hybrid capture methods

This application is a continuation-in-part application entitled "nucleic acid detection by target-specific hybrid Capture method" filed 10/20 2004 in 35 U.S. C. § 120, U.S. patent application Ser. No. 11/005,617 filed 12/6 2004, the application filed 10/20, the continuation-in-part application of U.S. patent application Ser. No. 10/971,251, which has been abandoned now, and the continuation-in-part application of U.S. patent application Ser. No. 10/971,251 filed 6/15/2000, all of which are incorporated herein by reference.

Technical Field

The present invention relates to the field of nucleic acid detection methods, and in particular to the detection of nucleic acids by target-specific hybrid capture methods.

Background

The detection of specific nucleic acid sequences present in biological samples is important for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic alterations associated with cancer, studying genetic susceptibility to disease, and measuring response to various types of treatments. Conventional techniques for detecting and quantifying specific nucleic acid sequences are nucleic acid hybridization and target amplification.

Various hybridization methods are available for the detection and study of nucleic acids. In conventional hybridization methods, the nucleic acids identified are either in solution or attached to a solid support. The nucleic acids are detected using labeled nucleic acid probes capable of hybridizing to the nucleic acids. Recently, new hybridization methods have been developed to improve the sensitivity and specificity of detection. One example is the Hybrid Capture described in U.S. application Ser. No. 07/792,585 and U.S. Pat. No. 6,228,578A method. Although these new hybridization methods provide a significant improvement over traditional methods, they still do not have the ability to completely distinguish between highly homologous nucleic acid sequences.

Polymerase Chain Reaction (PCR) is the most commonly used method for amplification of target nucleic acids. However, PCR is somewhat limited when a large number of different targets are amplified simultaneously (i.e., multiplex reactions), which may not only cause PCR artifacts (artifacts) such as primer dimers, but also spurious target amplification. In an attempt to overcome this limitation, universal primers may be used when multiple targets have regions of homology. However, homology between species is never one hundred percent, so primers will have several mismatches with different targets, causing non-uniform amplification. When different amounts of target are present in a sample, the amplification efficiency of different PCRs also varies, leading to non-uniform and non-specific amplification of different targets.

It is therefore an object of the present invention to provide a method for detecting a target nucleic acid sequence which not only provides improved speed and sensitivity, but is also highly specific and capable of discriminating between a plurality of highly homologous nucleic acid target sequences.

Disclosure of Invention

The present invention provides a novel nucleic acid detection method, referred to herein as target-specific hybrid capture ("TSHC"). TSHC is a rapid, highly specific and sensitive method that can distinguish and detect highly homologous nucleic acid target sequences.

One embodiment of the present invention relates to a method for the detection and/or quantification of one or more target nucleic acids for the rapid and sensitive detection of a target nucleic acid sequence, the method comprising the steps of targeted enrichment, amplification and detection.

In one embodiment of the method of the invention, one or more target nucleic acids are detected by: capturing a target nucleic acid on a solid support by mixing the target nucleic acid, a nucleic acid probe complementary to the target nucleic acid (one of which is RNA and the other is DNA), and the solid support; removing unbound target nucleic acid and nucleic acid probe; amplifying the captured target nucleic acid or nucleic acid probe to form a plurality of amplicons, wherein the presence of the amplicons indicates the presence of the target nucleic acid; and detecting the target nucleic acid by mixing the target nucleic acid, a selective and distinguishable oligonucleotide hybridizing to a portion of the target nucleic acid (i.e., capture sequence probe, CSP), and a nucleic acid probe complementary to the other portion of the target nucleic acid (i.e., signal sequence probe, SSP), wherein either the probe or target is RNA and the other is DNA, and detecting DNA: RNA hybrids via a DNA: RNA hybrid specific binding agent that is directly or indirectly labeled. The SSP is not limited solely as a means of generating a detection signal; capture can also be initiated by hybridization to a target nucleic acid using a DNA: RNA hybrid specific binding agent for the target enrichment step.

In another embodiment, the plurality of target nucleic acids are detected by: hybridizing a plurality of target nucleic acids to nucleic acid probes complementary to the target nucleic acids to form DNA-RNA hybrids; capturing said DNA RNA hybrid using a DNA RNA hybrid specific antibody bound to a solid support; removing unbound target nucleic acid and nucleic acid probe; amplifying the captured target nucleic acid or nucleic acid probe using random primers and a DNA polymerase to form a plurality of amplicons, wherein the presence of the plurality of amplicons indicates the presence of the target nucleic acid; hybridizing a nucleic acid probe complementary to a portion of the target nucleic acid sequence, thereby forming a DNA-RNA hybrid between the target and the probe; hybridizing an oligonucleotide bound to a solid support to the remainder of the target nucleic acid, wherein the solid support is selective; selecting said oligonucleotide complex; and detecting the plurality of target nucleic acids by binding a DNA: RNA hybrid specific binding agent to the DNA: RNA hybrid.

In another embodiment, one or more target DNAs are detected by a multiplex method comprising the steps of: hybridizing a plurality of target DNAs with RNA probes complementary to the target DNAs to form DNA-RNA hybrids; capturing the DNA RNA hybrid with a DNA RNA hybrid specific antibody bound to the bead; removing unbound nucleic acids and nucleic acid probes by washing excess nucleic acids and probes; isothermally amplifying the target DNA using random primers and a DNA polymerase to form a plurality of amplicons; hybridizing an RNA probe (i.e., SSP) complementary to a portion of the target DNA to form a DNA-RNA hybrid; hybridizing a specific DNA oligonucleotide to the other portion of the target DNA, wherein said DNA oligonucleotide is bound to a selective microbead; and detecting the plurality of target DNAs by binding detectably labeled DNA RNA hybrid specific antibodies to the DNA RNA hybrids and selecting the target DNAs using microbeads conjugated to selective oligonucleotides (i.e., CSPs), wherein the DNA RNA hybrid specific antibodies are detectably and distinguishably labeled. The presence of each target is detected by SSP using the labeled DNA: RNA antibodies, which form DNA: RNA hybrids with the target, while separating and selecting various targets based on microbeads bound with oligonucleotides (i.e., CSP). The presence of the amplicon and DNA RNA hybrid indicates the presence of the target DNA.

Detailed Description

The present invention provides methods for enriching, amplifying, and detecting the presence of one or more nucleic acids in a test sample. More specifically, the present invention provides highly specific and sensitive methods that are capable of distinguishing and detecting highly homologous nucleic acid sequences.

One embodiment of the present invention relates to a rapid and sensitive method for multiplex detection of target nucleic acid sequences, which can detect a plurality of different target nucleic acid sequences simultaneously. This method can be automated. The method mainly comprises three steps: target enrichment, target amplification and target detection, which provides a semi-quantitative or qualitative method for nucleic acid diagnosis. This embodiment of the invention can be used in multiplex mode for detection of target nucleic acid sequences in purified or unpurified samples, where the concentration of target can be as little as about 50 copies or less than about 100 copies of target per milliliter of sample, or 10 copies per milliliter⁸Multiple copies. Detection of a single pathogen or target in a biological sample, such as but not limited to HSV and HPV types, can be performed in a multiplex format, preferably but not limited to 50 plexuses (50-plexes).

It is an advantage of the present invention that such a multiplex method of detecting a plurality of target nucleic acid sequences is a relatively fast, sensitive and accurate method with two levels of specificity, which is an advantageous feature for clinical detection. By using target-specific sequences, the two levels of specificity are obtained in the steps of enrichment and detection of the target.

Target enrichment is the first step, which prepares the sample for amplification by separating non-specific nucleic acids and contaminants from specific targets. The presence of non-specific or unwanted nucleic acid sequences reduces the sensitivity of target amplification by interfering with amplification and detection. The target enrichment step purifies the sample from possible inhibitors and eliminates non-specific nucleic acids, thereby providing a first level of specificity. Purging non-specific nucleic acids allows for efficient amplification in the isothermal amplification step and thereby minimizes competition between cognate targets. Target enrichment also denatures double-stranded targets, producing single-stranded DNA for efficient amplification. To enhance capture of the target, various reagents can be used in the target enrichment step. For example, subtilisin may be used to improve target capture (especially in clinical samples of blood). Subtilisin or carbonyl hydrolase, an alkaline protease secreted by members of the genus bacillus. When the target nucleic acid is captured by the beads, the presence of subtilisin causes the beads to form a tight granular precipitate, thereby increasing the ease with which unbound or unwanted material can be eluted. In addition, the target enrichment step also concentrates the target nucleic acid into a small volume.

Enrichment of the target is achieved by mixing a target nucleic acid, a DNA: RNA hybrid specific binding agent, e.g., a nucleic acid probe complementary to the target nucleic acid, such as a signal sequence probe, and a solid support, wherein the target nucleic acid hybridizes to the DNA: RNA hybrid specific binding agent to form a DNA: RNA hybrid on the solid support, and removing any nucleic acid that does not form a DNA: RNA hybrid or is not captured by the solid support.

In particular, the desired separation of target and non-specific nucleic acids is achieved by forming DNA: RNA hybrids that are captured on a solid phase or solid support, such as paramagnetic microbeads modified with a DNA: RNA hybrid specific binding agent such as, but not limited to, a DNA: RNA hybrid specific antibody (e.g., hybrid capture antibody (HC-Ab)), a monoclonal or polyclonal antibody or fragment thereof, a protein, a catalytically inactive ribonuclease H, a nucleic acid aptamer, or an oligonucleotide with the ability to bind and form a triplex structure. Haruki et al reported that ribonuclease H was Mg-free²⁺And a catalyst, which binds to the DNA-RNA hybrid by surface plasmon resonance for the binding of Escherichia colise HI to RNA-DNA hybrids using surface plasmon resonance (kinetic and stoichiometric analysis of binding of e.coli ribonuclease HI and RNA-DNA hybrids) j.biol.chem.272: 22015-]. This step of target enrichment can be performed on any solid support, such as on a microplate, a microchip, a microbead, a paramagnetic/non-paramagnetic microbead or any of the solid phases mentioned previously. For example, target DNA, RNA probes or a collection of different RNA probes (if in a multiplex reaction) are mixed with microbeads conjugated with conjugated DNA: RNA HC-antibodies. The target DNA and the RNA probe form a DNA-RNA hybrid. RNA HC-antibody is bound to a solid support, such as paramagnetic beads. Once the DNA RNA hybrid is captured onto the solid support, any unbound nucleic acid sequences and contaminants are eluted, preferably by repeated washes using a buffer that does not reduce or affect the hybridization.

Alternatively or in addition to using DNA-RNA hybrid specific antibodies, oligonucleotides or polynucleotides of any length complementary to the target bound to a solid phase can be used to capture the target nucleic acid sequence. For example, oligonucleotides or probes that recognize specific HPV types may be bound to a solid phase, such as a plate, chip, or bead. Several microbeads conjugated with specific and known probes form a microbead assembly. For example, each microbead pool is specific for one HPV type. In a multiplex format, multiple bead sets identifying different HPV types may be used, one for each HPV type. Oligonucleotides useful for target Nucleic acid capture may be partially or wholly Locked Nucleic Acids (LNA), Peptide Nucleic Acids (PNA) or with other modifications [ Current Protocols in Nucleic acid chemistry, Eds. Serge L. Beaucage et al, John Wiley&Sons2004]. The nucleic acid probe can be up to about 100% of the length of the target nucleic acid and can range in length from about 50 bases to about 10,000 bases. The target nucleic acid can beDNA or RNA. If the target nucleic acid is RNA, cDNA can be generated from the RNA target by any known conventional method, or the target RNA can be captured by a DNA probe, wherein the hybridized DNA probe will be amplified and detected.

The target enrichment step is not limited by the examples set forth herein. One skilled in the art will understand how to purify a target nucleic acid in a sample to remove or eliminate contaminants, inhibitors, and other non-specific nucleic acids from the sample according to the principles set forth herein. Sample preparation is known and understood in the art by commercially available methods and kits, such as plasmid small or large scale preparation kits, gel recovery kits, and DNA and RNA purification kits. In certain cases where lower sensitivity is acceptable, these methods and kits can be used to facilitate the target enrichment step.

After target enrichment, the sample is subjected to nucleic acid amplification. Isothermal target amplification of nucleic acids using random sequence oligonucleotide primers and a DNA polymerase with strand displacement activity overcomes various limitations such as, but not including, the limited multiplexing capability and heterogeneous amplification of multiplex PCR. The amplified sequences or amplicons are identified by hybridization to sequence-specific oligonucleotides. Amplification can also be performed using a single target rather than multiplex format, and amplicons can be combined for hybridization assays.

Primer extension and activation strand displacement may be performed using a DNA polymerase. Alternatively, if a DNA probe is used to capture the target RNA, the hybridized DNA probe is amplified and detected. Another embodiment entails reverse transcribing the target RNA to produce cDNA as the DNA target, which can be captured as described above. This second step preferably uses isothermal amplification of the target nucleic acid. In particular, random sequence oligonucleotide primers and DNA polymerases with strand displacement activity may be useful in isothermal amplification. In one embodiment, a solid phase or solid support such as, but not limited to, magnetic microbeads having captured target nucleic acids can be used directly in the amplification mixture. Any DNA polymerase (including but not limited to phi29DNA polymerase, Bst DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, and DNA polymerase I) can be used to amplify the DNA target, the amplification being directed by random primers.

For optimal efficiency, random primers can have a particular ratio of dG, dC, dT and dA monomers. For optimal manipulation, one or two nucleotide bases may be omitted entirely. The pool of random primers need not include all possible sequences. The sequences included in the library need not be in equimolar concentrations. In fact, the pool of random primers may comprise a subset of primer sequences that are selective for a particular disease. The length may vary from about 4 to about 20 nucleotides, and preferably from about 5 to about 8 nucleotides. The length depends on the proportion of monomers in the primer. The optimal amplification temperature varies from about 25 ℃ to about 70 ℃, preferably from about 28 ℃ to about 40 ℃, which can be determined by one skilled in the art based on the length of the primers and does not require undue experimentation. The optimum temperature can be estimated by using commercially available software that predicts the annealing temperature of the oligonucleotide. An example of such commercially available software is OLIGO version 6.0 or 6.41^TM(Molecular biology instruments; Cascade, CO). For example, the target DNA may be amplified for 2 hours using a pentamer primer in combination with phi29DNA polymerase.

For example, pentamer primers are protected from exonuclease degradation by modification. The structure of the primer may include a phosphorothioate bond or a 2' -O-methyl group. Depending on the sensitivity required, the amplification reaction is preferably carried out for 1-3 hours, which is much shorter than the duration of the published protocol for the phi29DNA polymerase reaction [ Gary J.Vora et al, Applied and environmental microbiology, 70 (5): 3047-3054, 2004, incorporated by reference. The unique reagent formulation, in combination with the selected target enrichment step and target detection step, enables relatively short amplification times.

The target amplification step of the invention preferably utilizes isothermal amplification capable of achieving: amplification of equilibrium efficiency for an unlimited number of targets; amplification of the entire sequence, rather than of fragments as in the case of Polymerase Chain Reaction (PCR); and allows target detection even if a portion of the target sequence in the biological sample is removed. For example, the L1 region is frequently deleted in HPV, however, by using hybridization probes against the L1 region and the E region [ MH Einstein and GN Goldberg, Cancer invest.20: 1080-1085, 2002], the deleted sequence can still be detected at the same level of sensitivity, thus enabling detection of the target even if a portion of the sequence is absent. The multiplex isothermal amplification may be performed at a temperature ranging from about 25 ℃ to about 40 ℃ for a reaction time ranging from 1 hour to 3 hours. Non-limiting examples of isothermal amplification include rolling circle amplification [ Lizardi p., huang x., Zhu z., Bray-Ward p., Thomas d., Ward d. "Mutation detection and single molecule counting using isothermal rolling circle amplification" ("Mutation detection and single molecule counting using isothermal rolling circle amplification") nat. gene.1998, 19: 225-232), multiple displacement amplification [ Little M. et al, "strand displacement amplification and homology detection in a second generation probe system" ("Strand Displacement amplification and homology real-time detection introduced into second generation probe system") Clin. chem.1999, 45: 777- & 784] and protein-guided DNA amplification [ Blanco M., Lazaro J., de Vega M., Bonnin A., Salas M "Terminal protein-printed DNA amplification" "(Terminal protein-guided DNA amplification") Proc. Natl. Acad. Sci USA, 1994, 91: 12198-12202]. Other target amplification embodiments include those in which the target is released from the solid support prior to addition to the amplification mixture. Non-limiting methods for releasing the target nucleic acid include alkali treatment, high temperature incubation, and ribonuclease H treatment. The unique reagent formulation, in combination with the selected target enrichment step and target detection step, allows for the use of relatively short amplification times. Reagents for isothermal amplification (optionally including ribonuclease H) can be operated simultaneously with target amplification, which will increase the number of guide sites on the target DNA. These methods allow denaturation and solid support release, making target amplification more efficient.

The next step involves the detection of a single target nucleic acid sequence in the amplified nucleic acid. The sensitivity and specificity of this step is generally dependent on the amplification efficiency. Detection and specification of the target nucleic acid is performed by hybridizing different portions of the amplified target product or amplicon to a single capture sequence probe that is bound to a solid support and the specific probe is complementary to the target sequence to form a target complex. Hybridization of different portions of the target nucleic acid sequence to the two probes provides two levels of specificity, ensuring detection and identification of specific targets in the sample.

Oligonucleotide or capture sequence probes bound to a solid support, preferably selective microbeads, are useful in obtaining a level of specificity. Hybridization of the CSP to the amplified target nucleic acid, followed by selection of the microbeads, enables separation of the target complex from unbound or partially bound entities. CSP is usually added in excess to ensure binding. Preferably, the CSP and SSP have no overlapping sequences; however, it is possible to perform the method in an embodiment where the CSP and SSP have overlapping sequences that are complementary to the target nucleic acid sequence.

The Capture Sequence Probes (CSP) or oligonucleotides may be bound or attached to a solid phase, solid support or solid matrix, which comprises for example polystyrene, polyethylene, polypropylene, polycarbonate or any solid plastic material in the shape of plates, slides, dishes, microbeads, particles, cups, strips (strips), chips, ribbons (strips), microplates and microarrays. Solid phases also include glass beads, glass test tubes, and any other suitable glass product. Functionalized solid phases such as plastics or glasses which have been modified to contain carboxyl, amino, hydrazide, aldehyde groups, nucleic acids or nucleotide derivatives on the surface can also be used. Any solid phase such as plastic, metal, magnetic or glass microparticles, microbeads, ribbons, tubes, slides, strips, chips, microchips or microplates can be used.

The capture sequence probes or oligonucleotides are nucleic acid probes comprising at least 8 bases, preferably 15 to 100 bases, and more preferably 20 to 40 bases. The capture sequence probes are preferably identifiable, either by their known location on a solid support, such as a sub-well plate, or by a distinguishable colored dye, for example when bound to a microbead. Means for identifying such capture sequence probes are known in the art and are exemplified in the specification.

Hybridization with sequence-specific probes that can be attached to a solid support not only allows identification of specific targets, but also provides a second level of specificity to the assay, making it a more reliable clinical assay. If a lower sensitivity is desired, no signal amplification is required, but detection using (signal) oligonucleotides or polynucleotides linked to reporter genes (biotin, fluorophores, enzymes, etc.) is used. There may be more than 1 detector oligonucleotide per target. Both the capture and detection oligonucleotides may be additionally modified, such as PNA, LNA, etc. The length of the oligonucleotide or nucleic acid probe can range from about 15 bases to about 10,000 bases, or up to 100% of the target length.

The Signal Sequence Probe (SSP) may be unlabeled RNA to form a DNA: RNA hybrid that is recognized during the detection step by, for example, a labeled hybrid-specific binding agent. Since the SSP is not labeled, it does not generate background noise when not bound to the target, thereby resulting in higher specificity. For example, if the RNA signaling probe is 8,000 bases (Kb) in size and binds 20 bases per antibody, a signal amplified by about 400-fold can be obtained. As previously set forth, typically the signal sequence probe comprises at least 15 bases, but may be up to or greater than about 1000 bases, preferably between about 15 bases and 100 bases. Other non-limiting examples of signal sequence probes include labeled RNA probes and labeled DNA probes. Hybridization is not limited to non-overlapping regions, but may actually include overlapping regions. The RNA-DNA hybrid complexes bound to the solid support can be detected in a multiplex manner, for example using PE-labeled antibodies, carboxylated distinguishable microbeads, and flow cytometry.

One embodiment of target (or amplicon) detection utilizes a liquid-based array. Bead arrays are commercially available, and carboxylated polystyrene bead arrays are preferred in this example. For example, a 96-well plate has a bead pool mix in each well. There are 13 bead sets in the 13 clumps, each with a specific "signature", and the signature is provided by a dye inside each bead. The ratio of these dyes is specific for each bead set and can be distinguished between each bead set. Capture Sequence Probes (CSPs) or oligonucleotides specific for a target nucleic acid are applied or bound to a specific set of microbeads. When the target is hybridized to the CSP or oligonucleotide-bound microbeads, a specific set of microbeads is selected and then detected using complementary nucleic acid probes and a labeled DNA: RNA hybrid-specific binding agent. The selection or separation may be performed in a flow cytometer, in which the microbeads are advanced one by one through two lasers: one of which selects for the signature on the beads while the other detects the target identified by the labeled DNA: RNA hybrid-specific binding agent. In this way, multiple targets can be distinguished and detected. In addition, labeled DNA RNA reagents allow for enhanced detection of signals, thereby improving the specificity and sensitivity of the assay.

Another embodiment of the liquid-based bead array of the present invention utilizes a sub-well plate. A sub-well plate is a microplate having, for example, 96 main wells, wherein each individual main well is subdivided into a plurality of sub-wells. The use of such sub-well plates as platforms for multiplexing has been previously described [ a.roda, et al, clin.chem 46: 1654-1660, 2002, incorporated by reference. However, as used and illustrated previously, the use of sub-well platforms is limited due to the problem of "cross-talk" between different sub-wells. However, the modified sub-aperture platforms described herein substantially eliminate the cross-talk problem between different sub-apertures by using a mask. Each sub-well preferably has a known target-specific capture sequence probe attached to the well. A target nucleic acid can be added to the main well where it hybridizes to its complementary nucleic acid probe, i.e., a signal sequence probe. A DNA-RNA hybrid is formed between a portion of the target nucleic acid and the signal sequence probe. The capture sequence probes bind to different portions of the target nucleic acid, thereby capturing the DNA RNA hybrid to a solid phase. As previously set forth, the detection of the target nucleic acid is carried out using a complementary nucleic acid probe that hybridizes to the target nucleic acid and a labeled DNA: RNA hybrid-specific antibody, and by, for example, a known capture sequence probe. Both signal amplification and the use of a mask, which acts as a cover to eliminate cross-talk between sub-wells, enable the detection of specific and sensitive target nucleic acids with minimal background noise.

A wide variety of methods are available for detecting and identifying target sequences, including but not limited to: dot blot hybridization, reverse dot blot hybridization on nylon membranes, hybridization of slide arrays, chip arrays, bead arrays, arrays on the bottom of multi-well plates, and the like. Non-limiting examples of solid supports for hybridization include bead arrays (such as commercially available bead array products), slide arrays, plate arrays (such as arrays on the bottom of each well of a microplate), sub-well plates (microplates with 16-64 small sub-wells in each well), and electrode arrays such as the GenOHM system (GeneOhmSciences, Inc.). Drummond et al, Nature Biotech, 21: 1192-1199(2003). Different types of reporter genes can be applied to generate the detection signal by, for example, fluorescence, chemiluminescence, and gold nanoparticles. As previously discussed, the means of detection may include fluorescence, any enzyme-based signal, chemiluminescent or colorimetric signal, light scattering by gold particles, and the like.

In one embodiment, the reporter gene may be linked to a hybrid-specific binding agent, such as, but not limited to: DNA hybrid-specific antibody (HC-Ab), protein, catalytically inactive ribonuclease H, nucleic acid aptamer, or oligonucleotide having the ability to bind and form triplex structures. Each labeled binding agent binds to a nucleotide segment of a DNA: RNA hybrid, e.g., at least 20 nucleotides in length, thereby providing signal amplification.

Another embodiment of the present invention is for multiplex detection of multiple target DNAs in an unpurified sample, the protocol comprising the steps of: 1) denaturing the target DNA to produce a single-stranded target sequence; 2) adding an RNA probe and a magnetic microbead, wherein the microbead has been previously bound to a hybrid-specific antibody; 3) removing any unbound target and contaminants by washing; 4) subjecting the captured target to isothermal amplification in the presence of DNA polymerase and other reagents that initiate amplification and amplicon formation for a relatively short period of time, e.g., 1-3 hours; 4) denaturing the amplicons to produce single-stranded sequences; 5) the amplified target is hybridized with an oligonucleotide (capture probe) bound to a solid support, such as a microbead or microplate, and a signal sequence probe; and 6) detecting the presence or absence of each target DNA by discriminating the targets and detecting the signal sequence probe complex based on the characteristics of the capture probes.

In one embodiment of the invention, hybridization of the target and probe can be performed simultaneously with the capture step using the hybrid binding agent in the same mixture and at an elevated temperature. Elevated temperatures throughout the process may allow for increased specificity of target capture while reducing reaction time. It is understood that low, moderate, and high stringency hybridization/wash conditions can be varied using a variety of ingredients, buffers, and temperatures well known and practiced by those skilled in the art. For additional stringency conditions, see t.maniotis et al,molecular Cloning: a Laboratory Manual (molecular weight) And (2) Longding: laboratory manual)Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). Depending on the overall assay conditions, one-step hybridization and capture may also be more efficient than sequential hybridization and capture.

Methods for detecting a target nucleic acid of interest by target enrichment, target amplification and target detection in the presence of other nucleic acids provide rapid and sensitive methods for detecting multiple targets simultaneously in the same reaction sample. Such a method is useful in clinical diagnostic applications for identifying a variety of disease conditions, such as whether a patient is infected with Human Papillomavirus (HPV) and determining which specific HPV type the patient has, in order to better diagnose and treat the patient. Other diseases associated with specific nucleic acids are readily known in the art and can be identified using the detection methods described herein.

Any source of nucleic acid, in purified or unpurified form, can be used as a test sample. For example, the test sample may be a food or agricultural product, or a human or veterinary clinical specimen. Typically, the test sample is a biological fluid such as urine, blood, plasma, serum, saliva, and the like. Alternatively, the test sample may be a tissue sample suspected of carrying a nucleic acid of interest. The target nucleic acid in the test sample may initially be present as a discrete molecule, such that the sequence to be detected constitutes the entire nucleic acid, or may be only a component of a larger molecule. The nucleic acid sequence to be detected does not have to be present initially in pure form. The test sample may comprise a complex mixture of nucleic acids in which the target nucleic acid may correspond to a gene of interest contained in the entire human genomic DNA or RNA or a portion of the nucleic acid sequence of a pathogenic organism, which is a minor component of the clinical sample.

The target nucleic acid in the test sample can be DNA or RNA, such as messenger RNA, of any origin, including cells or tissues of bacteria, yeast, viruses, and higher organisms (e.g., plants or animals). The target may also be cDNA synthesized from RNA by reverse transcription. Methods for extracting and/or purifying such nucleic acids are well known in the art. The target nucleic acid may be double-stranded or single-stranded. In one embodiment of the method, the target nucleic acid is single-stranded, or made single-stranded by conventional denaturation techniques prior to the hybridization and amplification steps of the method. In one embodiment, base denaturation techniques or high temperatures are used to denature double stranded target DNA.

The term "oligonucleotide" as used herein refers to a nucleic acid molecule consisting of two or more deoxyribonucleotides or ribonucleotides. The desired oligonucleotide may be prepared by any suitable method, such as by molecular biological means from naturally occurring nucleic acids or by de novo synthesis. Non-limiting examples of oligonucleotides and nucleic acid probes are set forth herein.

Nucleic acid probes are nucleic acid sequences that hybridize to complementary RNA or DNA sequences in a test sample. Detection of the probe indicates the presence of the particular nucleic acid sequence in the test sample. The nucleic acid probe may be detected directly or indirectly. In one example, the target-specific hybrid capture method employs two types of nucleic acid sequence probes: capture Sequence Probe (CSP) and Signal Sequence Probe (SSP).

Capture sequence probes or oligonucleotides include nucleic acid sequences that: which is capable of hybridizing to a distinct region of the target nucleic acid and being captured to a solid phase or which is capable of capturing the target nucleic acid sequence. The CSP used in the detection method may be DNA, RNA, Peptide Nucleic Acid (PNA) or other nucleic acid analogues. PNAs are oligonucleotides in which the sugar-phosphate backbone is replaced by a polyamide or "pseudopeptide" backbone. In a preferred embodiment, the CSP is DNA. The CSP has a minimum length of at least 8 bases, preferably between 15 and 100 bases, and more preferably between 20 and 40 bases. The CSP is substantially complementary to the target nucleic acid sequence to which it is to hybridize. The sequence of the CSP is preferably at least 75% complementary to the target-hybridizing region, more preferably 100% complementary to the sequence. It is also preferred that the CSP comprise less than or equal to 75% sequence identity, more preferably less than 50% sequence identity, to undesired non-target sequences considered to be present in the test sample. The sequence in the target nucleic acid to which the CSP binds is preferably at least about 12 bases, more preferably 20-40 bases. The target nucleic acid sequence to which the CSP hybridizes is preferably a unique sequence or a cluster-specific sequence. A cluster-specific sequence is a plurality of related sequences that form discrete clusters. For example, CSP may comprise sequences that recognize specific human papillomavirus types, such as but not limited to HPV-16, HPV-18 and HPV-31.

In one embodiment, the CSP used in the detection method may comprise one or more modifications in the nucleic acid that allow specific capture of the probe onto a solid phase. For example, the CSP may be modified by tagging the CSP with at least one ligand by methods well known to those skilled in the art, including, for example, nick translation, chemical or photochemical incorporation. In addition, the CSP may be tagged with one or more types of tags at multiple locations. For example, CSP can be labeled with streptavidin-bound biotin, digoxigenin-bound anti-digoxigenin, or 2, 4-Dinitrophenol (DNP) bound anti-DNP. Fluorophores can also be used to modify the probes. Examples of fluorophores include fluorescein and derivatives, phycoerythrin, allophycocyanin, phycocyanin, rhodamine, texas red, or other proprietary fluorophores. The fluorophore is typically attached by chemical modification and is conjugated to a fluorophore-specific antibody, such as an anti-fluorescein. Those skilled in the art will understand that: CSP can also be tagged by incorporating modified bases comprising any chemical group recognized by a specific antibody. Other methods for tagging tags and nucleotide sequences for capture onto a solid phase coated with a substrate are well known to those skilled in the art. An overview of nucleic acid markers can be found in "DNA Diagnostics-Molecular Techniques and analysis" ("DNA Molecular Diagnostics and Automation") Science, 242: 229-237(1988), which is incorporated herein by reference. In another embodiment, the CSP is tagged with biotin at the 5 'and 3' ends of the nucleotide sequence. In another embodiment, the CSP is not modified, but is captured onto a solid phase substrate because the sequence comprised by the CSP is capable of hybridizing to the substrate.

The SSP used in the detection method may be DNA or RNA. The signal sequence probe comprises a nucleic acid sequence capable of hybridizing to a region of the target nucleic acid adjacent to the unique region recognized by the CSP. In a particular embodiment of the invention, the SSP and the target nucleic acid form a DNA: RNA hybrid. Thus, in this embodiment, if the target nucleic acid is DNA, then the preferred SSP is RNA. Similarly, if the target nucleic acid is RNA, then the preferred SSP is DNA. SSPs are typically at least 15 bases in length. However, SSPs can be up to or greater than 1000 bases in length. Longer SSPs are preferred. An SSP may comprise a single nucleic acid fragment, or a plurality of smaller nucleic acid fragments, each of which is preferably between 15 and 100 bases in length.

The sequences of the CSP and SSP are selected such that they do not hybridize to the same region of the target nucleic acid or to each other. To eliminate competition between CSP and SSP, the SSP sequence and CSP sequence corresponding to the region of the target nucleic acid sequence can be modified. For example, an SSP may have deletions not encompassed by a CSP. In addition, the CSP and SSP are selected to hybridize to regions in the target, e.g., 50,000 bases from each other. The distance between the sequence hybridizing to the CSP and the sequence hybridizing to the SSP in the target nucleic acid is preferably between about 1 to 50,000 bases. More preferably, the distance between the CSP and the SSP on the target nucleic acid is less than 3,000 bases, and most preferably, the distance is less than 1,000 bases.

In another embodiment, a portion of the SSPs used in the detection methods form DNA: RNA hybrids with a single-stranded target nucleic acid sequence, which hybrids are detected by a DNA: RNA hybrid specific binding agent, and another portion of the SSPs are capable of hybridizing to the target nucleic acid. The SSP may be prepared as follows: first a single-stranded DNA sequence complementary to a sequence in the target nucleic acid is cloned into a single-stranded DNA vector, and then RNA complementary to the DNA vector sequence is hybridized to produce a DNA RNA hybrid. For example, if M13 is used as a DNA vector, M13 RNA hybridizes to the M13 DNA sequence in the vector to produce a DNA: RNA hybrid. The SSP thus produced forms a DNA, an RNA hybrid portion and a single-stranded portion capable of hybridizing to sequences in the target nucleic acid. The single-stranded DNA should be at least 10 bases in length and may be up to or greater than 1000 bases in length. Alternatively, the DNA: RNA hybrid portion of the SSP can be formed during or after the reaction in which the single-stranded portion of the SSP hybridizes to the target nucleic acid. SSPs can be linear, circular, or a combination of two or more of these forms. The DNA RNA hybrid portion of the SSP provides an amplified signal for detection of captured hybrids using, for example, DNA RNA hybrid specific antibodies labeled or recognized by a labeled entity as set forth herein.

In another embodiment, the SSP used in the detection method is a molecule that does not contain a sequence capable of hybridizing to the target nucleic acid. In this example, a bridge probe (bridge probe) is used, which comprises a sequence capable of hybridizing to the target nucleic acid and a sequence capable of hybridizing to the SSP. The bridge probe may be DNA, RNA, Peptide Nucleic Acid (PNA) or other nucleic acid analogues.

In another embodiment, a portion of the SSP forms a DNA: RNA hybrid with the complementary nucleic acid probe, and the single-stranded portion of the SSP comprises a sequence complementary to a sequence in the bridge probe. A bridge probe capable of hybridizing to both the target nucleic acid and the SSP is used as an intermediate for ligating the SSP to the target nucleic acid. DNA RNA hybrids are detected by a DNA RNA hybrid specific binding agent that can be labeled or detected by a labeling entity. The CSP hybridizes to different portions of the target nucleic acid sequence, thereby capturing the target, bridge probe, and DNA: RNA hybridization complex onto a solid phase or solid support. SSP may be prepared as set forth above.

In another embodiment, the SSP used in a target nucleic acid detection method comprises a plurality of sets of repeat sequences that are complementary to single-stranded RNA sequences capable of hybridizing to a bridge probe. DNA oligonucleotide probes containing sequences complementary to the repeat sequences can be used to hybridize with SSPs to produce DNA-RNA duplexes required for signal amplification.

In another embodiment, the bridge probe comprises a poly (a) tail in addition to a sequence capable of hybridizing to the target nucleic acid. The SSP used in this example comprises a poly (dT) DNA sequence. Thus, the bridge probe is able to hybridize to the SSP through its poly (a) tail. RNA probes containing poly (A) sequences can be used to hybridize to the poly (dT) DNA sequences remaining in the SSP to form DNA: RNA hybrids. SSP containing poly (dT) sequences and RNA probes containing poly (A) sequences are preferably 100 to 5,000 bases in length.

The SSP used in the methods of detecting a target nucleic acid sequence of the present invention may be unmodified or modified, as may the CSP using the methods set forth above and/or methods known in the art. In a preferred embodiment, the SSP is a probe that is not covalently modified.

It should be understood that multiple CSPs and/or SSPs may be employed in the detection method of the present invention.

In another embodiment, oligonucleotide probes comprising complementary sequences of two or more different regions of a target nucleic acid are fused together and used as capture sequence probes in the methods of the invention. Alternatively, a single probe can be designed and generated that contains sequences complementary to a single or multiple target nucleic acids. This type of probe is also referred to herein as a "fused" CSP. The fusion capture sequence probe was as effective as the combination of two unfused CSPs when used at the same concentration.

The nucleic acid probes of the invention may be produced by any suitable method known In the art, including, for example, chemical synthesis, isolation from naturally occurring sources, recombinant production, and asymmetric PCR (McCabe, 1990 In: PCR Protocols: A guide to methods and applications (In PCR Protocols: methods and application guidelines), San Diego, CA., Academic Press, 76-83). As in the case of preparing an oligonucleotide microarray on a microchip, it may be preferable to chemically synthesize probes using one or more segments and then ligating the segments. Narang et al (1979meth. Enzymol.68: 90), Brown et al (1979meth. Enzymol.68: 109) and Caruthers et al (1985 meth.Enzymol 154: 287), all of which are incorporated herein by reference, describe several chemical synthesis methods. Alternatively, cloning methods may provide convenient nucleic acid fragments that can be isolated for use as promoter primers. Prior to hybridization with the target nucleic acid, the double-stranded DNA probe is first rendered single-stranded using, for example, conventional denaturation methods.

Hybridization is performed under standard hybridization conditions well known to those skilled in the art. The conditions under which a probe hybridizes to a nucleic acid sequence vary from probe to probe, depending on a number of factors, such as the length of the probe, the number of G and C nucleotides in the sequence, and the composition of the buffer used for the hybridization reaction. Moderately stringent hybridization conditions, generally known to those skilled in the art, are: a condition of about 25 ℃ below the melting temperature of a double-stranded DNA with complete base pairing. Generally, higher specificity is obtained by using incubation conditions with higher temperatures, in other words more stringent conditions. Well-known test manual by Sambrook et al, MOLECULAR CLONING: the hybridization conditions for oligonucleotide probes are described in great detail in Chapter 11 of A LABORATORY MANUAL, molecular cloning: A LABORATORY Manual, second edition, Cold Spring Harbor LABORATORY Press, New York (1990), incorporated herein by reference, including a description of the factors involved and the level of stringency necessary to ensure specific hybridization. Typically, hybridization is performed in aqueous buffer, and the conditions for such hybridization, such as temperature, salt concentration, and pH, are selected to provide sufficient stringency so that the probes specifically hybridize to their respective target nucleic acid sequences but not to any other sequences.

In general, the efficiency of hybridization between the probe and target is improved under the following conditions: the amount of probe added is in molar excess with respect to the template, preferably 2 to 10⁶Molar excess, more preferably 10³To 10⁶Molar excess. For efficient capture, each CSP is provided at a concentration in the final hybridization solution of at least 25fmole/ml (25pM), preferably between 25fmole/ml and 10 fmole/ml⁴fmole/ml (10 nM). The concentration of each SSP in the final hybridization solution is at least 15ng/ml, preferably 150 ng/ml. Table A shows the conversion of SSP concentration to molarity expressed in ng/ml.

TABLE A

In the detection step, hybridization of the CSP and the SSP to the target nucleic acid may be performed simultaneously or sequentially in either order. In one embodiment, the hybridization of the CSP to the target nucleic acid and the hybridization of the SSP to the target nucleic acid are performed simultaneously. In one embodiment, the resulting DNA: RNA hybrid can then be captured onto a solid phase. The solid phase may be coated with a substrate that specifically binds to a ligand attached to the CSP. A portion of the target nucleic acid sequence and the SSP form a DNA: RNA hybrid, while another portion of the target nucleic acid sequence hybridizes to the CSP attached to the solid phase. In another embodiment, hybridization of the SSP to the target nucleic acid is performed after hybridization of the CSP to the target nucleic acid. In this case, the CSP may be immobilized on a solid phase before or after hybridization. Both the CSP and the target nucleic acid sequence can be bound to a solid phase during the SSP hybridization reaction.

It will be understood by those skilled in the art that solid phases, solid supports or solid matrices can be used interchangeably and include, for example, glass, silicon, metal, nitrocellulose, polystyrene, polyethylene, polypropylene, polycarbonate or any solid plastic material in the shape of plates, slides, dishes, microbeads (microbeads), particles, microparticles, cups, tubes, slides, strips, chips, microchips, ribbons, membranes, microplates with sub-wells and microarrays. Solid phases also include glass beads, glass test tubes, and any other suitable glass product. Functionalized solid phases such as plastics or glasses that have been modified so that their surfaces contain carboxyl, amino, hydrazide, aldehyde, nucleic acid or nucleotide derivatives may also be used.

In one embodiment, the CSP is labeled with biotin and streptavidin-coated or avidin-coated solid phase is applied to capture the hybrids. In another embodiment, a streptavidin-coated microplate or microplate is used. These plates may be coated passively or covalently. Another embodiment uses a microplate in which each individual well has several sub-wells, and CSPs are attached individually to each sub-well. Another embodiment applies the CSP bound to the microbeads by any known and commonly used binding method, such as the use of spacers or linkers. When the CSP is bound to a solid support, the CSP can be used to capture and/or select target nucleic acids complementary thereto.

The captured DNA RNA hybrid can be detected by conventional means well known in the art. DNA RNA hybrids can be detected directly or indirectly using labeled or distinguishable DNA RNA hybrid specific binding agents, such as labeled monoclonal or polyclonal antibodies or fragments thereof specific for DNA RNA hybrids, antibodies specific for one or more ligands attached to the SSP, labeled antibodies, or modifications detectable by the SSP itself.

A preferred method detects the captured hybrids by using DNA: RNA hybrid specific antibodies. In this embodiment, the DNA: RNA hybrid-specific antibody is preferably labeled with an enzyme, a fluorescent molecule, or a biotin-avidin species, and is preferably non-radioactive. The label may be detected directly or indirectly by conventional means known in the art, such as a colorimeter, photometer or fluorescence detector. One preferred label is, for example, alkaline phosphatase. Other labels known to those skilled in the art may also be used as a means of detecting the bound double stranded hybrid. Detection is carried out by methods conventionally used in the art, for example, as described in Coutlee et al, j.clin.microbiol.27: the colorimetric or chemiluminescent methods described in 1002-1007(1989) are incorporated by reference. Preferably, the bound alkaline phosphatase is detected by chemiluminescence by adding a substrate that can be activated by alkaline phosphatase. Chemiluminescent substrates activated by alkaline phosphatase are well known in the art.

Detection of the captured DNA: RNA hybrids is preferably accomplished by binding a bound DNA: RNA hybrid-specific binding agent, such as, but not limited to, a DNA: RNA hybrid-specific antibody, to the DNA: RNA hybrids during the incubation step. In one embodiment, the DNA: RNA hybrid specific antibody is bound to a solid phase, such as a paramagnetic microbead. The DNA: RNA hybrids are captured by placing paramagnetic microbeads bound to antibodies specific for the DNA: RNA hybrids in a magnetic field force, and then the surface is washed to remove any excess antibodies and other unhybridized material. This step enriches or purifies the target nucleic acid sequence. These target enrichment techniques are known in the art. For example, manual washing may be performed using a repeat pipette or syringe, a simple pump regulated by a disintegration controller, or gravity flow through a reservoir with attached tubing. Commercially available and effective pipe wash systems may also be used.

Non-limiting examples of capture sequence probes useful in the present invention include those having sequences for HSV-1 and HSV-2. CSPs against HPV include those with SEQ ID NO: 1-64 CSPs. HPV types encompassed by these sequences include: HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82, several of which are high-risk HPV.

In another embodiment, the method employs blocker probes in addition to the CSP and SSP. The blocker probe comprises a sequence substantially complementary to the sequence of the CSP. The sequence of the blocker probe is preferably at least about 75% complementary to the sequence of the CSP, more preferably 100% complementary to the CSP. The addition of the blocker probe to the hybridization reaction mixture inhibits the hybridization of unhybridized CSP to the cross-reactive nucleic acid sequences present in the target and thus improves the specificity of the detection. Typically, the blocker probe is at least about 5 bases to about 12 bases. In one embodiment of the invention, the concentration of blocker probe in the hybridization reaction is preferably in excess of the concentrations of CSP and SSP. Preferably, the blocker probe is present in about a 2-fold molar excess, although it may be present in up to a 10,000-fold molar excess. Blocker probes may be DNA, RNA, peptides, non-specific nucleic acids (PNA) or other nucleic acid analogs.

In one embodiment, blocker probes complementary to full-length or nearly full-length CSPs are used. Following the reaction to form a hybrid between the CSP, SSP and target nucleic acid, one or more blocker probes may be added to the reaction and allowed to hybridize for a desired time. The hybridization product is then detected as set forth above.

In another embodiment, blocker probes complementary to only a portion of the CSP and shorter than the CSP are used. These blocker probes have lower melting temperatures than CSP. Preferably, the melting temperature of the blocker probe is 10 degrees lower than the melting temperature of the CSP. In this embodiment, the blocker probe is preferably added to the target nucleic acid simultaneously with the CSP and SSP. Since the blocker probe has a lower melting temperature than the CSP, the onset temperature of hybridization is chosen so that the blocker probe does not interfere with the hybridization of the CSP to its target sequence. However, when the temperature of the hybridization mixture is adjusted to be lower than the temperature for target hybridization, the blocker probe hybridizes to the CSP and effectively prevents hybridization of the CSP to the cross-reactive nucleic acid sequence. For example, in a hybrid capture process, a blocker probe may be added when the hybrid product is incubated on a streptavidin-coated microplate at room temperature. Non-limiting examples of blocker probes are shown in table 2.

The following examples illustrate the amplification methods and detection assays of the invention and the use of the kits. These examples are provided only for illustrating and explaining the present invention and should not limit the scope of the present invention in any way. All references set forth herein are expressly incorporated by reference in their entirety into this specification.

Example 1

Target Specific Hybrid Capture (TSHC) assay protocol

Known concentrations of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) virus particles (Advanced biotechnology, inc., Columbia, MD) or clinical samples were diluted using negative control medium (Digene Corp., Gaithersburg, MD) or negative neck samples (Digene Corp). Various dilutions were prepared and aliquoted into individual microfuge tubes (microfuge tubes). Half the volume of denaturing agent 5100-0431(DigeneCorp.) was added. The test sample was incubated at 65 ℃ for 45 minutes to denature the nucleic acids in the sample.

After denaturation, a hybridization solution containing Signal Sequence Probes (SSP) (600 ng/ml each) and Capture Sequence Probes (CSP) (2.5 pmole/ml each) was added to the samples and incubated at 74 ℃ for 1 hour. A blocker probe solution containing one volume of 4x probe dilution (Digene Corp.), one volume of denaturing agent and two volumes of negative control medium was then added to the hybridization mixture and incubated at 74 ℃ for 15 minutes.

In a second series of experiments, after nucleic acid denaturation, a hybridization solution containing SSP (600 ng/ml each), CSP (2.5 pmole/ml each) and blocker probe (250 pmole/ml each) was added to the samples and incubated at 74 ℃ for 1 hour.

The tubes containing the reaction mixture were cooled to room temperature for 5 minutes, and an aliquot was removed from each tube and transferred to a single well of a 96-well streptavidin capture plate (Digene Corp.). The plates were shaken at 1100rpm for 1 hour at room temperature. The supernatant was then decanted and the plate was washed twice with SNM wash buffer (Digene Corp.) and briefly inverted to remove residual wash buffer. Alkaline phosphatase anti-RNA/DNA antibody DR-1 reagent (Digene Corp.) was then added to each well and incubated at room temperature for 30 minutes. Each well was then washed a number of times, including the steps of: 1) washed 3 times with Sharp wash buffer (Digene Corp.) at room temperature; 2) incubating the plate with Sharp wash buffer for 10 min at 60 ℃ on a heat block (heat block); 3) washed twice with Sharp wash buffer at room temperature; 4) washed once with SNM wash buffer (Digene Corp.) at room temperature. After removal of the residual liquid, chemiluminescent substrates 5100-0350(Digene Corp.) were added to each well and incubated for 15 minutes at room temperature. Individual wells were then read on a flat panel luminometer to obtain Relative Light Unit (RLU) signals.

Solutions containing negative control media or known HSV negative neck specimens were used as negative controls for the test samples. The signal-to-noise ratio (S/N) was calculated as the ratio of the mean RLU obtained in the test samples to the mean RLU of the negative control. The signal-to-noise ratio is used as the basis for determining the capture efficiency and detection of target nucleic acids. S/N values of 2 or more were arbitrarily assigned as positive signals, while S/N values less than 2 were considered negative. The Coefficient of Variation (CV) was calculated by taking the standard deviation of duplicate runs, dividing by the mean and multiplying by 100 to obtain the percentage value that measures the variability of the run in a sample set.

The capture sequence probes and blocker probes used in the assay were synthesized using the method described by Cook et al (1988 Nucl. acid.Res., 16: 4077-95). Unless otherwise indicated, the capture sequence probes used in the experiments set forth herein were biotinylated at the 5 'and 3' ends.

The signal sequence probes used in the assays were RNA probes, which were prepared using the method described by Yiserail et al (1989, Methods in EnzymoL, 180: 42-50).

All CSPs were tested to determine their type-specific capability in the type-specific hybrid capture set forth herein. The CSPs of SEQ ID NO: 36-56 do not exhibit cross-reactivity characteristics with other types and can be used in assays.

Table 1: HPV capture sequence probes

Probe needle	Sequence of	Size (bp)	HPV type and sequence position
				ZL-1	GTACAGATGGTACCGGGGTTGTAGAAGTATCTG[SEQ ID NO：1]；	33	HPV165360-5392
ZL-4	CTGCAACAAGACATACATCGACCGGTCCACC[SEQ ID NO：2]	31	HPV16495-525
				DP-1	GAAGTAGGTGAGGCTGCATGTGAAGTGGTAG[SEQ ID NO：3]	31	HPV165285-5315
DP-4	CAGCTCTGTGCATAACTGTGGTAACTTTCTGGG[SEQ ID NO：4]	33	HPV16128-160
				SH-1	GAGGTCTTCTCCAACATGCTATGCAACGTCCTG[SEQ ID NO：5]	33	HPV31505-537
SH-4	GTGTAGGTGCATGCTCTATAGGTACATCAGGCC[SEQ ID NO：6]	33	HPV315387-5419
				VS-1	CAATGCCGAGCTTAGTTCATGCAATTTCCGAGG[SEQ ID NO：7]	33	HPV31132-164
VS-4	GAAGTAGTAGTTGCAGACGCCCCTAAAGGTTGC[SEQ ID NO：8]	33	HPV315175-5207

AH-1	GAACGCGATGGTACAGGCACTGCAGGGTCC[SEQ ID NO：9]	30	HPV185308-5337
				AH-2	GAACGCGATGGTACAGGCACTGCA[SEQ ID NO：10]	24	HPV185314-5337
AL-1	ACGCCCACCCAATGGAATGTACCC[SEQ ID NO：11]	24	HPV184451-4474
				PA-4	TCTGCGTCGTTGGAGTCGTTCCTGTCGTGCTC[SEQ ID NO：12]	32	HPV18535-566
18-1AB	*(TTATTATTA)CTACATACATTGCCGCCATGTTCGCCA[SEQ ID NO：13]	36	HPV181369-1395
				18-2AB	(TTATTATTA)TGTTGCCCTCTGTGCCCCCGTTGTCTATAGCCTCCGT[SEQ ID NO：14]	46	HPV181406-1442
18-3AB	(TTATTATTA)GGAGCAGTGCCCAAAAGATTAAAGTTTGC[SEQ ID NO：15]	38	HPV187524-7552
				18-4AB	(TTATTATTA)CACGGTGCTGGAATACGGTGAGGGGG TG[SEQ ID NO：16]	37	HPV183485-3512
18-5AB	(TTATTATTA)ACGCCCACCCAATGGAATGTACCC[SEQ ID NO：17]	33	HPV184451-4474
				18-6AB	(TTATTATTA)ATAGTATTGTGGTGTGTTTCTCACAT[SEQ ID NO：18]	35	HPV1881-106
18-7AB	(TTATTATTA)GTTGGAGTCGTTCCTGTCGTG[SEQ ID NO：19]	30	HPV18538-558
				18-8AB	(TTATTATTA)CGGAATTTCATTTTGGGGCTCT[SEQ ID NO：20]	31	HPV18634-655

PE-1	GCTCGAAGGTCGTCTGCTGAGCTTTCTACTACT[SEQ ID NO：21]	33	HPV18811-843
				PZ-2	GCGCCATCCTGTAATGCACTTTTCCACAAAGC[SEQ ID NO：22]	32	HPV4577-108
PZ-5	TAGTGCTAGGTGTAGTGGACGCAGGAGGTGG[SEQ ID NO：23]	31	HPV455295-5325
				CS-1	GGTCACAACATGTATTACACTGCCCTCGGTAC[SEQ ID NO：24]	32	HPV45500-531
CS-4	CCTACGTCTGCGAAGTCTTTCTTGCCGTGCC[SEQ ID NO：25]	31	HPV45533-563
				PF-1	CTGCATTGTCACTACTATCCCCACCACTACTTTG[SEQ ID NO：26]	34	HPV451406-1439
PF-4	CCACAAGGCACATTCATACATACACGCACGCA[SEQ ID NO：27]	32	HPV457243-7274
				PA-1	GTTCTAAGGTCCTCTGCCGAGCTCTCTACTGTA[SEQ ID NO：28]	33	HPV45811-843
45-5AB	(TTATTATTA)TGCGGTTTTGGGGGTCGACGTGGAGGC[SEQ ID NO：29]	36	HPV453444-3470
				45-6AB	(TTATTATTA)AGACCTGCCCCCTAAGGGTACATAGCC[SEQ ID NO：30]	36	HPV454443-4469
45-8AB	(TTATTATTA)CAGCATTGCAGCCTTTTTGTTACTTGCTTGTAATAGCTCC[SEQ ID NO：31]	49	HPV451477-1516
				45-9AB	(TTATTATTA)ATCCTGTAATGCACTTTTCCACAAA[SEQ ID NO：32]	34	HPV4579-103

45-10AB	(TTATTATTA)GCCTGGTCACAACATGTATTAC[SEQ ID NO：33]	31	HPV45514-535
				45-11AB	(TTATTATTA)CAGGATCTAATTCATTCTGAGGTT[SEQ ID NO：34]	33	HPV45633-656
ON-1	TGCGGTTTTGGGGGTCGACGTGGAGGC[SEQ ID NO：35]	27	HPV453444-3470
				ZZ-1	GGCGCAACCACATAACACACAGAACCACAAAAC[SEQ ID NO：36]	33	HPV185285-5315
DXA-1	GTTCTACACGGGTTTGCAGCACGATCAACAACG[SEQ ID NO：37]	33	HPV331175-1207
				PRA-1	CGCTGCTTGTGGTGGTCGGTTATCGTTGTCTG[SEQ ID NO：38]	32	HPV333389-3420
TT-1	GACGTAGTGTCGCCTCACATTTACAACAGGAC[SEQ ID NO：39]	32	HPV35732-763
				TT-7	CTCGCTTGGTGGGGTTGTAGGGGAGCTCGG[SEQ ID NO：40]	30	HPV353432-3461
NH-1	GCTGTAGTTGTCGCAGAGTATCCCGTGAGG[SEQ ID NO：41]	30	HPV39833-862
				FT-7	GTGAGCCTGTGTTATATGTAGTGCCCGAATCCC[SEQ ID NO：42]	33	HPV395358-5390
SHA-4	CCACCTCCTGCGTCCACTACACCTAGCACTA[SEQ ID NO：43]	31	HPV455295-5325
				SHA-1	TGCGTGCGTGTATGTATGAATGTGCCTTGTGG[SEQ ID NO：44]	32	HPV457243-7274

OCA-1	AATTAGCGCATTGCCCCGTCCAACGTCCCG[SEQ ID NO：45]	30	HPV51482-511
				TAA-7	CGCCGTGCACGTGTAGCCACCATATTTAATCAC[SEQ ID NO：46]	33	HPV514124-4156
LFA-1	CGAATTGTGTGAGGTGCTGGAAGAATCGGTGC[SEQ ID NO：47]	32	HPV52140-171
				PTA-7	GATCGTTCACAACTTTTACCTGCACCGGATCC[SEQ ID NO：48]	32	HPV525066-5097
ZOA-1	CTAGGTTCTCTAGATGTTTGTCTCCAGCACCCC[SEQ ID NO：49]	33	HPV56521-553
				NAA-4	CTGTCGGTATTGTCTGTGTCGCTGATGTGTG[SEQ ID NO：50]	31	HPV563543-3573
LA-1	GATACACACACATTTGCAGCCCGGTCCACACA[SEQ ID NO：51]	32	HPV581180-1211
				LA-7	GGTGGCAAAGGACGTATGTGAGTGCAGAGGAC[SEQ ID NO：52]	32	HPV585376-5407
ZV-4	GCGTTGCGGAGGGGTATGATAGTTGCTCAGAAG[SEQ ID NO：53]	33	HPV593385-3417
				ZV-10	GTCTAGGCGTGTAGGAGGAAACAAGATGGGG[SEQ ID NO：54]	31	HPV597543-7573
PNA-1	CTGAACACAGCAGTTCTCTATACCAATGGCGCTATTTC[SEQ ID NO：55]	38	HPV6881-118
				PNA-4	TTGGTTGCCCCTGAGCAGTCGGACCCTATGGATA[SEQ ID NO：56]	34	HPV685211-5244

CDA-4	GCGCCGCATTGCTGCACCTCGTTTATATAGCAGGGCATTTTC[SEQ ID NO：57]	42	HPV824827-4868
				CDA-11	CCTGGCGCATGTCATACACACCACATTACTC[SEQ ID NO：58]	31	HPV827596-7626
CTA-1	CACGAAGTGTCAGTGCACAGTATGCCTTGC[SEQ ID NO：59]	30	HPV736006-6045
				CTA-16	GCCATGTACTTCACAAACTGTTAATACTGGTGATTGTCCC[SEQ ID NO：60]	40	HPV73720-749
DLA-14	CCTACACAGTACAAGTGGAGGCCATCACCCG[SEQ ID NO：61]	31	HPV263566-3596
				RBA-16	GTCTGACACATACTGTTGTAACCCATAGTTAAACACAGG[SEQ ID NO：62]	39	HPV267759-7797
BNA-1	GCTGTCTCCCTGTCTTCCTGTGTATTGTTTAT AAGTGTATT[SEQ ID NO：63]	41	HPV661051-1092
				DLA-32	GTACAGCACTAAAATGGAGTTTGGTGTGTTACTATAGTGCATAC[SEQ ID NO：64]	44	HPV667420-7464

The sequences in parentheses are "tail" sequences not indicated by HSV

Table 2: HPV blocker probes

Probe needle	Sequence of	Size and breadth	In combination with CSP
				PA-6	ACTCCAACGACGCAGA[SEQ ID NO：65]	16	PA-4
ZZ-2	(TTTTGTGGTTCTGTGTG[SEQ ID NO：66]	17	ZZ-1
				ZZ-3	TTATGTGGTTGCGC[SEQ ID NO：67]	14	ZZ-1
DXA-2	CGTTGTTGATCGTGC[SEQ ID NO：68]	15	DXA-1
				DXA-3	TGCAAACCCGTGTAG[SEQ ID NO：69]	15	DXA-1

PRA-2	CAGACAACGATAACCG[SEQ ID NO：70]	16	PRA-1
				PRA-3	ACCACCACAAGCAGC[SEQ ID NO：71]	15	PRA-1
TT-2	GTCCTGTTGTAAATGTG[SEQ ID NO：72]	17	TT-1
				TT-3	AGGCGACACTACGTC[SEQ ID NO：73]	15	TT-1
TT-8	CGAGCTCCCCTACAA[SEQ ID NO：74]	15	TT-7
				TT-9	CCCCACCAAGCGA[SEQ ID NO：75]	13	TT-7
NH-2	CCTCACGGGATACTC[SEQ ID NO：76]	15	NH-1
				NH-3	TGCGACAACTACAGC[SEQ ID NO：77]	15	NH-1
FT-8	GGATTCGGGCACTA[SEQ ID NO：78]	14	FT-7
				FT-9	CATATAACACAGGCTCAC[SEQ ID NO：79]	18	FT-7
SHA-5	TAGTGCTAGGTGTAGTGG[SEQ ID NO：80]	18	SHA-4
				SHA-6	ACGCAGGAGGTGG[SEQ ID NO：81]	13	SHA-4
SHA-2	TACATACACGCACGCA[SEQ ID NO：82]	16	SHA-1
				SHA-3	CCACAAGGCACATTCA[SEQ ID NO：83]	16	SHA-1
TAA-8	GCTACACGTGCACGGCG[SEQ ID NO：84]	17	TAA-7
				TAA-9	GTGATTAAATATGGTGG[SEQ ID NO：85]	17	TAA-7
OCA-2	GGGACGTTGGACG[SEQ ID NO：86]	13	OCA-1
				OCA-3	GGCAATGCGCTAAT[SEQ ID NO：87]	14	OCA-1
LFA-2	CACCGATTCTTCCAG[SEQ ID NO：88]	15	LFA-1
				LFA-3	CACCTCACACAATTCG[SEQ ID NO：89]	16	LFA-1
PTA-8	GGATCCGGTGCAG[SEQ ID NO：90]	13	PTA-7
				PTA-9	GTAAAAGTTGTGAACGATC[SEQ ID NO：91]	19	PTA-7
ZOA-2	GGTGCTGGAGACAA[SEQ ID NO：92]	14	ZOA-1
				ZOA-3	ACATCTAGAGAACCTAG[SEQ ID NO：93]	17	ZOA-1

NAA-5	CACACATCAGCGACA[SEQ ID NO：94]	15	NAA-4
				NAA-6	CAGACAATACCGACAG[SEQ ID NO：95]	16	NAA-4
LA-2	TGTGTGGACCGGG[SEQ ID NO：96]	13	LA-1
				LA-3	CTGCAAATGTGTGTGTAT[SEQ ID NO：97]	18	LA-1
LA-8	GTCCTCTGCACTCACAT[SEQ ID NO：98]	17	LA-7
				LA-9	ACGTCCTTTGCCAC[SEQ ID NO：99]	14	LA-7
ZV-5	TTCTGAGCAACTATCATAC[SEQ ID NO：100]	19	ZV-4
				ZV-6	CCCTCCGCAACG[SEQ ID NO：101]	12	ZV-4
ZV-11	CCCCATCTTGTTTCC[SEQ ID NO：102]	15	ZV-10
				ZV-12	TCCTACACGCCTAGAC[SEQ ID NO：103]	16	ZV-10
PNA-2	TATAGAGAACTGCTGTGTTC[SEQ ID NO：104]	20	PNA-1
				PNA-3	GAAATAGCGCCATTG[SEQ ID NO：105]	15	PNA-1
PNA-5	CTCAGGGGCAACCA[SEQ ID NO：106]	14	PNA-4
				PNA-6	ATCCATAGGGTCCGAC[SEQ ID NO：107]	16	PNA-4
CDA-5	CAATGCGGCGC[SEQID NO：108]	11	CDA-4
				CDA-6	TATAAAC GAGGTGCAG[SEQ ID NO：109]	16	CDA-4
CDA-7	GAAAATGCCCTGCTA[SEQ ID NO：110]	15	CDA-4
				CDA-12	ACATGCGCCAGG[SEQ ID NO：111]	12	CDA-11
CDA-13	GAGTAATGTGGTGTGTATG[SEQ ID NO：112]	19	CDA-11
				CTA-2	GCAAGGCATACTGTG[SEQ ID NO：113]	15	CTA-1
CTA-3	CACTGACACTTCGTG[SEQ ID NO：114]	15	CTA-1
				CTA-17	GGACAATCACCAGTATTA[SEQ ID NO：115]	18	CTA-16
CTA-18	AGTTTGTGAAGTACATGG[SEQ ID NO：116]	18	CTA-16
				DLA-15	CGGGTGATGGCC[SEQ ID NO：117]	12	DLA-14

DLA-16	CCACTTGTACTGTGTAGG[SEQ ID NO：118]	18	DLA-14
				RBA-17	CTGTGTTTA ACT ATGGGT[SEQ ID NO：119]	18	RBA-16
RBA-18	TACAACAGTATGTGTCAGAC[SEQ ID NO：120]	20	RBA-16
				BNA-2	AAGACAGGGAGACAGC[SEQ ID NO：121]	16	BNA-1
BNA-3	CTTATAAACAATACACAGG[SEQ ID NO：122]	19	BNA-1
				DLA-33	ATGCACTATAGTAACACACC[SEQ ID NO：123]	20	DLA-32
DLA-34	ACTCCATTTTAGTGCTGTA[SEQ ID NO：124]	19	DLA-32

Example 2

Effect of distance between target sites of CSP and SSP on Capture efficiency

The effect of the distance between the hybridization sites of the Capture Sequence Probe (CSP) and the Signal Sequence Probe (SSP) on the HSV-1 target nucleic acid on capture efficiency was evaluated. CSPs were determined which hybridized to HSV-1 nucleic acid sequences 0.2kb, 3kb, 18kb, 36kb and 46kb from the SSP hybridization site. The general TSHC method set forth in example 1 was applied. The capture efficiencies were 100%, 50%, 30%, 19% and 7%, respectively (table 3). A steady decrease in relative capture efficiency was observed when the distance was increased from 0.2kb to 46 kb.

Table 3: effect of distance between target sites on Capture efficiency

CSP	SSP	Distance between target sites	Relative capture efficiency
BRH 19	H19	0.2Kb	100％
				F15R	H19	3Kb	50％
F6R	RH5B	18Kb	30％
				F15R	RH5B	36Kb	19％
F6R	H19	46Kb	7％

Example 3

Capture efficiency of various CSPs and SSPs in TSHC detection of HSV-1

In HSV-1 detection with TSHC, the Capture Sequence Probe (CSP) was targeted to four HSV-1 specific Signal Sequence Probes (SSPs): the capture efficiency of each of H19, RH5B, RH3, and R10 was evaluated. The criteria used to design capture sequence probes were: 1) in the HSV-1 nucleic acid sequence, the hybridization site of the CSP is within 1kb, preferably within 0.5kb, of the 5 'or 3' end of the hybridization site of the SSP; and 2) CSP comprises sequences unique to HSV-1 without stretches (stretches) of HSV-2 homologous sequences greater than 10 bases. The CSPs are designed to target the 5 'and 3' regions adjacent to the SSP hybridization site, each SSP preferably carrying a 5 'CSP and a 3' CSP. Commercially available OMIGA software (Oxford Molecular Group; Campbell, Calif.) was used as a means to identify these sites. The CSP is designed to have a melting temperature (Tm) between 70 ℃ and 85 ℃. The general TSHC method set forth in example 1 was applied. 11 CSPs of H19 (bound at 6 different sites), 6 CSPs of RH5B (bound at 3 distinct sites), 6 CSPs of RH3 (bound at 6 distinct sites) and 2 CSPs of R10 were tested. As shown in Table 4, efficient capture sequence probes for signal sequence probes H19, RH5B, and R10 were found.

Table 4: TSHC-detected CSP and SSP of HSV-1

Example 4

Clinical sample testing

Capture 2(hc 2) Using TSHC and hybridization^TM(ii) a Digene Corp.) two methods tested 64 clinical specimen plates (clinical specimen panels) for HSV-1 and HSV-2. The plate included 15 samples containing a known quantity of HSV-1 or HSV-2 and 49 samples known to be negative for HSV-1 and HSV-2 by PCR detection. Thus, 15 positive samples were "expected" to be positive in both HC2 and TSHC assays, and 49 negative samples were "expected" to be negative in both HC2 and TSHC assays.

The general TSHC method set forth in example 1 was applied. Tables 5 and 6 show the results using the HC2 method and the TSHC method, respectively. Using the HC2 method, 5 samples tested positive and 44 samples tested positive out of 49 samples "expected" to produce a negative result. By comparison, all 49 samples tested negative using the TSHC method. Therefore, the TSHC method is superior to the HC2 method in specificity in the detection of HSV-1 and HSV-2.

Table 5: observed and expected results of HC2 testing for HSV-1 and HSV-2

Table 6: observed and expected results for TSHC detection of HSV1 and HSV2

Example 5

Effect of combining probes in TSHC detection of HSV

The effect of the combination of the HSV-1 specific signal sequence probe and the capture sequence probe set on HSV-1 detection was evaluated. Two different sets of RNA signal sequence probe/biotinylated capture sequence probe combinations (group 1: H19 plus HZ-1; and group 2: RH5b plus TS-1 and TS-2) were used, respectively, for HSV-1 and HSV-2 cross-reactive TSHC detection. TSHC was also performed using a combination of RNA signal sequence probe/biotinylated capture sequence probe sets to evaluate the effect of the combination of the two probe sets on sensitivity and cross-reactivity. The general TSHC method set forth in example 1 was applied. The results shown in Table 7 clearly demonstrate that the additive effect of combining the two probe sets on the HSV-1 assay has no significant increase in HSV-2 cross-reactivity.

Table 7: sensitivity enhancement by combining HSV-1 specific CSP and SSP

Capture sequence probes	Signal sequence probe	VP/ml	RLU	CV	S/N
						HZ-1HZ-1HZ-1HZ-1	H19H19H19H19	010^5 HSV-110^6 HSV-110^7 HSV-2	60267231649	3％4％6％2％	1.04.538.90.8
TS-1，TS-2TS-1，TS-2TS-1，TS-2TS-1，TS-2	RH5BRH5BRH5BRH5B	010^5 HSV-110^6 HSV-110^7 HSV-2	78291236875	6％6％11％11％	1.03.830.61.0
						HZ-1，TS-1，TS-2HZ-1，TS-1，TS-2HZ-1，TS-1，TS-2HZ-1，TS-1，TS-2	H19，RH5BH19，RH5BH19，RH5BH19，RH5B	010^5 HSV-110^6 HSV-110^7 HSV-2	70457426367	12％10％1％6％	1.06.560.91.0

Example 6

Detection of HPV types in multiplex format

Rapid, sensitive and specific detection of 13 sequences representing different types of Human Papillomaviruses (HPV) in a single biological sample is performed in a multiplex format. This assay can analyze up to 96 samples in a 96 well microplate format over a 5 hour period. At least 500 copies of one type of virus can be detected within 5 hours after the start of the assay.

Protein G-paramagnetic microbeads (Dynal Corp.; Brown der, Wis.) and100^TMcarboxylated microbeads (Luminex-corp., Austin, TX) were used as solid carriers. Generally as understood in the art and Ausubel et al [ Current Protocols in Molecular Biology, New York, Wiley Publishing, 1993, incorporated by reference]Described, HPV RNA is prepared by in vitro transcription of HPV plasmids. The new experiment had three steps, including: 1) target enrichment; 2) target amplification; and 3) target detection.

Target enrichment:

samples for amplification are prepared by separating preselected specific targets from unwanted or non-specific DNA and contaminants. This step removes unwanted DNA, the presence of which greatly reduces the sensitivity of target amplification. By combining sequence specific RNA:the DNA hybrids were captured onto paramagnetic microbeads for target enrichment. The beads were initially modified with RNA: DNA hybrid specific antibodies or hybrid capture antibodies (Digene, Corp.). More specifically, HC-Ab paramagnetic microbeads were prepared by: 1mL of protein G paramagnetic beads (2.7X 10)⁹micro-bead/mL; dynal, Corp.) and 375. mu.g of HC-Ab were mixed and incubated in PBS for 40 minutes at room temperature to form bead-antibody (bead-Ab) complexes. The bead-Ab complexes were concentrated at the bottom of the wells by placing the plate on a magnetic grid (Dynal, Corp.) and washing once with triethanolamine at 8.2 at 0.2M, pH. The antibody was cross-linked to protein G in 20mM DMP/0.2M triethanolamine, pH 8.2 reagent for 30 minutes. The bead-Ab complex was washed 3 times with 1mL of PBS-0.05% Tween 20. The microbeads were then resuspended in 1mL of PBS-Tween and stored at 4 ℃.

Samples were serially diluted with various HPV plasmids (10kb) prepared in deionized water containing 100. mu.g/ml herring sperm DNA, 1M guanidine-HCl, 10mM Tris-HCl (pH 8.0), 10mM EDTA, 0.05% sodium azide. Each diluted sample (50. mu.l) was mixed in a 96-well microplate containing 25. mu.l of 1.75M NaOH per well and incubated at 50 ℃ for 15 minutes to denature the sample. A mixture (cocktail) of 13 types of HPV RNA (15 ng each per run) selected from Table 1 was added to 25 μ L of the denatured sample in a neutralization solution: 0.125M sodium citrate, 0.125M sodium phosphate, 0.6M triethanolamine, 0.6BES, 0.83M glacial acetic acid, 3% polyacrylic acid, 5mM EDTA, 0.05% sodium azide and 0.4% Tween-20(pH 3.6-4.1). Paramagnetic microbeads (5.4X 10 per run) bound to hybrid capture antibody (Hc-Ab) were then immediately added to the neutralized samples⁶One bead). HPV RNA and paramagnetic bead-antibody were hybridized with sample target sequences at 65 ℃ for 30 minutes with shaking at 1100 rpm. The magnetic microbead-target complexes were concentrated at the bottom of the wells by placing the plate on a magnetic grid (Dynal, Corp.). The supernatant was aspirated by pipette and the beads were washed 3 times with wash buffer (40mM Tris pH 8.2, 100mM sodium chloride, 0.05% sodium azide and 0.05% tween-20) to remove any contaminants.

Target amplification:

isothermal amplification of sample target DNA is performed using random primers and a DNA polymerase with strand displacement activity. The DNA target was amplified using phi29DNA polymerase, primed by random pentamers. The pentamer primers were synthesized with two 3' terminal phosphorothioate linkages to prevent nuclease degradation. Other modifications may include, but are not limited to, 2' O-methyl incorporated into the primer structure. The target DNA was amplified for 2 hours. The paramagnetic microbeads with the captured target nucleic acid obtained in the target enrichment step were resuspended in 20 μ l of a reaction consisting of: 50mM Tris-HCl, 10mM MgCl, pH7.5₂、10mM(NH₄)₂SO₄4mM DTT, 200. mu.g/ml acetylated BSA, 400. mu.M of each dNTP, 125. mu.M random pentamer and 1U phi29DNA polymerase (New England Biolabs, Inc).

Primers that may be useful in the amplification step are listed below.

Probe HPV 165 '- \ FAM \ TCAGGACCCACAGGAGCGACCCAG-3' \ BHQ

(SEQ ID NO：125)

Forward primer 5'-GCACCAAAAGAGAACTGCAATGT-3' (SEQ ID NO: 126)

Reverse primer 5'-CATATACCTCACGTCGCAGTAACT-3' (SEQ ID NO: 127)

Double-labeled (duel-labeled) probes and primers (Integrated DNA Technologies, Inc.; Salt LakeCity, UT)

FAM-fluorescent reporter gene; BHQ-Black hole quencher

Target detection

The amplified sequences were detected using a liquid-based bead array system that integrates optics, fluidics, and signal processing to enable multiplexing (Luminex technology). The Luminex technology uses a highly sensitive assay system based on fluorescent microbeads and reporter molecules that can detect multiple targets in the same assay well (see U.S. patent nos. 5,981,180, 6,524,793, 5,736,330, 6,449,562, 6,592,822, 6,632,526, 6,514,295, 6,599,331, 6,046,807. The Luminex detection equipment uses two lasers: one for detecting the fluorescent microbead itself and the other for the fluorescent reporter gene. The double-stained fluorescent microbeads are capable of multiplex detection of up to about 100 different targets. In this example, Luminex technology was used for HPV typing. This approach improves the sensitivity and robustness of the platform.

Using the manufacturer's protocol, two oligonucleotide sequences complementary to the late (L) and early (E) regions of specific HPV types [ MH Einstein and GN Goldberg, Cancer invest.20: 1080-. The sequence of the capture oligonucleotides is shown in table 1. Two oligonucleotides of a specific HPV type were attached to the same microbead set, eliminating false negative results caused by deletion of the HPV L-region that may occur in cancer cells. Since the hybridization is already performed in the same tube that retains the paramagnetic microbeads introduced during the first target enrichment step, no purification step of the amplicons is required before the detection step.

The detection step of the assay starts with denaturation of the amplicons for 15 minutes at 70 ℃ in 75. mu.l 438mM NaOH. A mixture of 13 different types of HPV RNA (15 ng each per assay) was added to 25ul of denatured sample solution: 0.125M sodium citrate, 0.125M sodium phosphate, 0.6M triethanolamine, 0.6BES, 0.83M glacial acetic acid, 3% polyacrylic acid, 5mM EDTA, 0.05% sodium azide and 0.4% Tween-20(pH 3.6-4.1). 13 types of oligonucleotides were then bound individually to carboxylated Luminex polystyrene microbeads (5X 10 per run)³One bead) and added immediately to the neutralized sample. RNA and bead-oligonucleotide were hybridized to the target at 65 ℃ for 30 minutes with shaking at 1100 rpm. The sample was then passed through a 96-well filter plate(Whatman) filtration and resuspension of the microbeads in 100. mu.l of 1 XPhosphate buffered saline (PBS) containing: 0.05% Tween-20, 10% goat serum and 10ng phycoerythrin-labeled mouse monoclonal DNA RNA hybrid specific antibody (PE-HC-Ab). The samples were incubated at room temperature (18-25 ℃) for 30 minutes with shaking at 1100 rpm. Excess antibody was removed by vacuum filtration and the microbeads with target complex were resuspended in 100 μ L of PBS containing 0.05% Tween-20. The samples were then analyzed by Luminex flow cytometer after adjusting the photomultiplier gain to 700 volts.

The increased sensitivity of this step is due to the long RNA signal sequence probe (approximately 6500bp in length) which allows binding of multiple phycoerythrin antibodies (PE-Ab), thus resulting in approximately 500 times signal amplification compared to other detection techniques such as methods using fluorophores bound to short hybridization probes. The length of the RNA signal sequence probe can range from about 500 bases to about 10,000 bases, or up to just less than 100% of the length of the target nucleic acid.

The results shown in table 8 show the sensitivity of HPV16 detection in the presence of all 13 HPV type microbead sets. As shown by the robust signal-to-noise ratio: the sensitivity of plasmid detection was greater than 500 copies per experiment. The starting sample volume is 50 μ l, but can be increased to 500 μ l or even the whole sample (for tube-based assays, not shown), which will improve assay sensitivity to an even higher degree. The results also show the specificity of HPV16 detection. Essentially, no HPV target-specific signal other than HPV16 was detected on the microbead set. The signal of table 8 (average, n ═ 4 replicates,% CV between 3% and 31%) is the Median Fluorescence Intensity (MFI) of 100 microbeads (events) per sample counted by flow cytometry. The background or noise value is the MFI of the sample without target.

Table 9 shows the sensitivity and specificity of detection of all 13 HPV types. Target HPV types are listed along the X-axis and nucleic acid probes are listed along the y-axis. The same HPV target and HPV probe for each type are highlighted in the diagonal of the table (positive results). These panels show a high level of specificity of the probe for its HPV target. This assay determines that the probes are specific for the corresponding HPV type targets. The results in table 9 indicate that e.g. the HPV16 target binds only to the corresponding HPV16 probe (positive result) and not to probes of other HPV types (negative result).

Table 8: sensitivity of HPV16 detection

Table 9: specificity of HPV types

Example 7

Detection of HPV16, HPV58 and HPV33

Neck samples infected with multiple types of HPV were modeled by mixing 3 plasmids each inserted with a different type of HPV sequence (10kb) in the sample buffer shown in example 6. This example shows the ability of the assay to detect multiple infections when more than 1 HPV type is present in the sample.

Assays for multiple target samples were performed as described in example 12. The sample is composed of 1X 10²Copy of HPV16, 1X 10⁴Of copyingHPV58 and 1X 10⁶Copy HPV 33. The results in fig. 7 show that even in the presence of other HPV types of high abundance, a low abundance HPV16 target was detected. Fig. 7 shows that HPV16 has a low signal-to-noise ratio, where the signal (average, n-4 replicates,% CV (coefficient of variation) between 3% and 31%) is the Median Fluorescence Intensity (MFI) of 100 microbeads (events) per sample counted by flow cytometry. The noise value is the MFI of the sample without the target plasmid.

Example 8

Detection of HPV in clinical samples

HPV was detected in neck samples collected in sample transport medium (STM; Digene Corp., Gaithersburg, Md) and initially used commercially available HC2High-Risk HPV DNA Test^TMassay (Digene Corp.) screening for high risk HPV types. The results of the HC2 test were compared to the results of the HPV typing test described herein.

Neck samples were collected using a brush and stored in storage medium (1M guanidine-HCl, 10mM Tris-HCl (pH 8.0), 10mM EDTA, and 0.05% sodium azide in deionized water) until use. Aliquots (50 μ l) of the neck samples were analyzed according to the manufacturer's recommendations either by HC2(Digene Corp) or by the method set forth in example 6. To compare the sensitivity of the two assays, positive samples were serially diluted into neck samples without HPV infection.

Table 10: comparison of clinical sample detection sensitivity

Positive sample diluent	HC2, Signal/cutoff	Typing test, S/N
			Undiluted	187.2	585.1
1∶10	21.2	310.4
			1∶100	2.3	199.2
1∶1000	＜1	113.4
			1∶10,000	＜1	43.1

The sensitivity of HC2 for clinical samples is reported in table 10 and shows a signal/cut-off of 2.3 corresponding to approximately 10,000 HPV targets/assay (100-fold diluted samples). Thus, the present invention is at least 100 times higher and equal to 100 copies/assay when compared to the sensitivity of the HPV typing assay of the present invention, with a signal-to-noise ratio (S/N) of about 43.

Example 9

Detection of HPV DNA in circular, linear and integrated form

The assay of the invention can detect linear and circular DNA sequences. Thus, amplification in the assay of the invention need not be by the "rolling circle" phenomenon. Therefore, the target of the assay of the invention need not be circular. They may be linear, because circular pathogenic DNA is nicked by denaturation, or because the pathogenic (viral) DNA target is integrated into the linear host (human) genome, or because the pathogenic DNA is a linear genome.

Samples including serial dilutions of either linear or circular HPV16 plasmid or DNA purified from CaSki cells were diluted in TE buffer. Linear plasmids were generated by restriction enzyme digestion of circular plasmids using standard techniques. The CaSki cell DNA is human genomic DNA that contains 500 copies of the HPV16 viral genome integrated per human genome. The concentration of HPV16 copies per test CaSki cell DNA was calculated based on the size and ratio of both human and HPV genomes and the optical density measurements of the purified CaSkiDNA.

Immediately prior to amplification, the target was heat denatured in the presence of random primers for 3 minutes at 95 ℃ in the following solution: 50mM Tris-HCl (pH 8.0), 0.5mM EDTA and 50. mu.M random hexamer primer (3' end bond is phosphorothioate). The single stranded target was then amplified for 120 min at 30 ℃ in 20. mu.l of a reaction consisting of: 50mM Tris-HCl (pH 7.5), 10mM MgCl₂、10mM(NH₄)₂SO₄4mM DTT, 0.2mg/ml acetylated BSA, 400. mu.M of each dNTP, 0.13mM random hexamer and 10U phi29DNA polymerase (Epicentre, Inc).

The amplified target was detected as set forth in example 6, except as follows. The RNA: DNA hybrid complexes were first tagged with a mouse monoclonal anti-RNA: DNA primary antibody (10. mu.g/assay) and then with a goat anti-mouse IgG secondary antibody conjugated to phycoerythrin (1.6. mu.g/assay).

The results in table 11 show that the assay of the invention detects circular, linear and integrated DNA with robust signal-to-noise ratios, thus demonstrating that the effectiveness of the assay is independent of the "rolling circle" mechanism. However, since amplification of circular targets is more efficient than amplification of linear targets, a "rolling circle" mechanism can accelerate the reaction.

Table 11: detection of circular, linear and integrated targets using the proposed method

The signal is the average of the Median Fluorescence Intensity (MFI) of 100 microbeads (events) per sample counted by flow cytometry (n-2). The noise value is the mean MFI of samples without target plasmid.

Example 10

Isothermal amplification catalyzed by PHI29DNA polymerase using random oligonucleotides

Another embodiment of the target amplification step uses random primers of modified oligonucleotides optimized for length. Random pentamer 5 '-npnpnpnpsnpsn-3' was prepared by IDT, Inc. To obtain the data generated in this example, the HPV16 plasmid was amplified in a total reaction volume of 20. mu.l by isothermal amplification at 30 ℃ for 2 hours. The DNA target was heat denatured at 95 ℃ for 3 minutes in the following 10. mu.l solution: 50mM Tris-HCl (pH 8.0), 0.5mM EDTA, 50. mu.M primer. Isothermal amplification was performed in the following 20. mu.l solution at 30 ℃ for 2 hours: 50mM Tris-HCl (pH 7.5), 10mM MgCl₂、10mM(NH₄)₂SO₄4mM DTT, 200. mu.g/ml acetylated BSA, 400. mu.M of each dNTP, 125. mu.M random pentamer and 1U phi29DNA polymerase (NewEngland Biolabs, Inc). After amplification, half the volume (10 μ l) of amplicons were detected in a Luminex flow cytometer system using the "primary/secondary antibody" detection protocol set forth in example 11.

Table 12: random DNA pentamers are the optimal primer length. All primers had two phosphorothioate modifications at the 3' end.

Copying/testing	5-mer	6-mer	7-mer	8-mer	9-mer	10-mer
							0	10	9	7	13	9	14
1×102	5873	990	16	10	17	12
							1×104	9864	5766	1469	176	36	26
1×106	12257	7567	7187	5444	2421	1621

Value ═ Luminex mean MFI

Table 13: comparison of random primers of different lengths and modifications

Value ═ Luminex mean MFI

Tables 12 and 13 show that random DNA hexamer primers with one phosphorothioate modification are optimal, and random DNA pentamer primers with two phosphorothioate modifications are optimal.

Example 11

Different methods for detecting amplified targets

Three different protocols for the detection of amplified targets are compared. One method utilizes biotinylated probes, which are deoxyoligonucleotides labeled with biotin at the 5' end. By Integrated DNATechnology inc; these oligonucleotides were prepared by chemical synthesis of Salt Lake City, UT. The other two methods used a labeled primary anti-HC-Ab (Digene core), and sequentially a primary anti-HC-Ab and a labeled secondary antibody (Digene core), which represent two embodiments of the present invention. The combination of flow cytometry detection and signal amplification of antibody-labeled or stained microbeads results in enhanced signal and thereby increases the sensitivity of the assay by more than 10-fold. Signal amplification, just as with the presence of macrocomplexes on the microbeads, generally enhances the background of the assay, thereby reducing or eliminating sensitivity. However, the method of the invention as set forth provides enhanced signal and improved sensitivity. The macrocomplexes on the beads can interfere with detection patterns in the Luminex flow cytometer system that depend on a certain size of the bead. The method of the present invention overcomes both of these problems.

A. In one method, biotinylated probes complementary to the target nucleic acid are applied to streptavidin (SA-PE; Pierce) that binds phycoerythrin (phycothithrin). The amplified HPV16 plasmid was denatured in 75. mu.l 438mM NaOH at 70 ℃ for 15 minutes. Two 5 ' biotinylated probes (5 ' -biotin-TATTTTATATGACACAATGT-3 ' (SEQ ID NO: 128); 5 ' -biotin-GGTTTGTGCTAACAATAAATGTATCCATAG-3 ' (SEQ ID NO: 129)) were each added (2 nanograms) to 25. mu.l of the following solutions of the denatured target nucleic acid sample: 0.125M sodium citrate, 0.125M sodium phosphate, 0.6M triethanolamine, 0.6BES, 0.83M glacial acetic acid, 3% polyacrylic acid, 5mM EDTA, 0.05% sodium azide and 0.4% Tween-20(pH 3.6-4.1). Approximately 1000 polystyrene microbeads were conjugated to oligonucleotides specific for the L and E regions of HPV16 (see Table 1) and immediately added to the neutralized sample. Hybridization was carried out at 50 ℃ for 30 minutes while shaking at 1100 rpm. 100 μ l of the following solution containing SA-PE (100ng) was added for each reaction: 438mM NaOH, 0.125M sodium citrate, 0.125M sodium phosphate, 0.6M triethanolamine, 0.6BES, 0.83M glacial acetic acid, 3% polyacrylic acid, 5mM EDTA, 0.05% sodium azide, and 0.4% Tween-20, and incubated at room temperature for 5 minutes. The sample is then transferred to a filter pad where unbound material is removed by vacuum filtration. The microbeads were resuspended in 100. mu.l of PBS-0.05% Tween-20 and analyzed by Luminex flow cytometer after adjusting the photomultiplier gain to 700 volts.

B. In the second method, the amplified HPV16 plasmid was denatured in 75. mu.l 438mM NaOH at 70 ℃ for 15 minutes. A mixture of 13 different types of HPV RNA (15 ng each per assay, obtained as described in example 6) was added to 25. mu.l of the following solutions of the denatured samples: 0.125M sodium citrate, 0.125M sodium phosphate, 0.6M triethanolamine, 0.6BES, 0.83M glacial acetic acid, 3% polyacrylic acid, 5mM EDTA, 0.05% sodium azide and 0.4% Tween-20(pH 3.6-4.1). 13 sets of oligonucleotide-conjugated polystyrene microbeads (5X 10 per run)³Beads) were added immediately to the neutralized sample. Each set of microbeads has two oligonucleotide sequences complementary to the late (L) and early (E) regions of a specific HPV type. The sequence of the capture oligonucleotides is shown in table 1. RNA and bead-oligonucleotide and target nucleic acid at 1100rpm shaking, 65 degrees C hybrid 30 minutes. The samples were then filtered in 96-well filter plates (Whatman) and 100. mu.l of PBS-0.05% Tween-20 containing 1. mu.g of primary anti-HC-Ab was added. After shaking for 5 minutes at room temperature, the reaction was filtered and 100. mu.l PBS (0.05% Tween-20 and 10% goat serum solution) containing 8. mu.g of PE-labeled secondary antibody (goat-anti mouse) was added. Incubation was continued for 20 minutes while shaking at room temperature. Excess antibody was removed by vacuum filtration and the microbeads with target complex were resuspended in 100. mu.l PBS (containing 0.05% Tween-20). The samples were then analyzed by Luminex flow cytometer after adjusting the photomultiplier gain to 700 volts.

C. In the third method, the amplified HPV16 plasmid was denatured in 75. mu.l 438mM NaOH at 70 ℃ for 15 minutes. A mixture of 13 different types of HPV RNA (15 ng each per assay, obtained as described in example 6) was added to 25. mu.l of the following solutions of the denatured samples: 0.125M sodium citrate, 0.125M sodium phosphate, 0.6M triethanolamine, 0.6BES, 0.83M glacial acetic acid, 3% polyacrylic acid, 5mM EDTA, 0.05% sodium azide and 0.4% Tween-20(pH 3.6-4.1). 13 sets of oligonucleotide-conjugated polystyrene microbeads (5X 10 per run)³Beads) were added immediately to the neutralized sample. Each set of microbeads has two oligonucleotide sequences complementary to the late (L) and early (E) regions of a specific HPV type. The sequence of the capture oligonucleotides is shown in table 1. RNA and bead-oligomer were hybridized to the target for 30 min at 1100rpm shaking at 65 ℃. The samples were then filtered through 96-well filter plates (Whatman) and the microbeads were resuspended in 100 μ l PBS containing: 0.05% Tween-20, 10% goat serum and 10ng mouse monoclonal RNA labeled with phycoerythrin DNA-specific antibody (PE-HC-Ab). The samples were then incubated at room temperature (18 ℃ -25 ℃) for 30 minutes with shaking at 1100 rpm. Excess antibody was removed by vacuum filtration and the microbeads with target complex were resuspended in 100. mu.l of PBS containing 0.05% Tween-20. The samples were then analyzed by Luminex flow cytometer after adjusting the photomultiplier gain to 700 volts.

Isothermal amplification of the HPV16 target was performed as described in example 6. The amplified target is detected using one of the three protocols set forth in scheme A, B or C above. The results are shown in table 14. Of the three protocols illustrated, detection with biotinylated probes (protocol a) involves the least time to carry out the method. However, this detection method also produces the lowest signal strength. The detection scheme using labeled primary antibody achieved maximum signal intensity and signal-to-noise ratio (Table 14), which achieved HC-Ab signal amplification (schemes B and C). Scheme C uses one less step than scheme B and is preferred.

Table 14: comparison of HPV16 detection after isothermal amplification

Example 12

HPV typing in 96-well NUCLEOLINK plates

In an alternative embodiment, a microplate may be used as a solid support for sequence detection in place of microbeads. A single HPV type-specific oligonucleotide is bound to the bottom of the well. The target nucleic acid is hybridized to the capture oligonucleotide and then detected by chemiluminescence.

In NucleoLink^TMIn the preparation of multiwell plates (Nalge Nunc International; New York), two oligonucleotide sequences complementary to the late (L) and early (E) regions of specific HPV types were bound to a Nucleolink plate using the protocol set out below. The sequence of the capture oligonucleotides is shown in table 1. By placing two oligonucleotides in each well, false negative results caused by deletion of HPV L regions commonly found in cancer cells can be eliminated. Type-specific capture oligonucleotides (two per HPV type) containing 5' -C12-amino linker were prepared at a final concentration of 100nM (0.1 pmole/. mu.L) in freshly prepared 10mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution at pH7 containing 10mM 1-methylimidazole. This solution was added to a Nucleolink 96 well plate at 100. mu.L per well (one type per well), the plate was sealed with a thermostable tape and incubated for 4 hours at 50 ℃. The solution was then decanted and the wells washed three times, soaked for 5 minutes and all washed three times at room temperature with 100mM Tris-HCl pH7.5 containing 150mM sodium chloride and 0.1% Tween-20. The wells were then washed three times, soaked for 5 minutes and washed three times with molecular biological grade water. The plates were then allowed to dry at room temperature for 30 minutes. The plates were stored in vinyl bags with desiccant at 4 ℃ until needed.

After storage, the NucleoLink plates with covalently bound oligonucleotides were removed to room temperature. Each well of the plate was blocked with 290. mu.l of 6% casein in 100mM Tris-HCl solution (pH7.2) containing 0.05% sodium azide at room temperature for 30 minutes. A standard sample (100pg/mL) of HPV plasmid (10kb) dilutions was prepared in deionized water solution consisting of: 100. mu.g/mL herring sperm DNA, 1M guanidine-HCl, 10mM Tris-HCl (pH 8.0), 10mM EDTA, 0.05% sodium azide. Samples (50. mu.l) were mixed with 25. mu.L of 1.75M NaOH in 96-well microplates and incubated for 30 min at 70 ℃. The following solutions of unlabeled RNA probes (15 ng per assay) were added to each well of the hybridization plate: 0.125M sodium citrate, 0.125M sodium phosphate, 0.6M triethanolamine, 0.6N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, 0.83M glacial acetic acid, 3% polyacrylic acid, 5mM EDTA, 0.05% sodium azide, and 0.4% Tween-20(pH 3.6-4.1). The solution became clear or yellowish. The contents of each well were transferred to their respective wells on a Nucleolink plate. The plate was incubated at 65 ℃ for 1 hour with shaking at 1150 rpm. The plate was cooled to room temperature and the solution decanted. To each well was added an alkaline phosphatase solution (100. mu.L/well) to which a mouse monoclonal anti-RNA: DNA antibody was bound and a 30% blocking solution (100 mM Tris-HCl pH7.2 containing 6% casein and 0.05% sodium azide) and the plate was incubated at 37 ℃ for 30 minutes, the alkaline phosphatase solution being: 0.1mM Tris-pH 7.4, 0.6M sodium chloride, 0.1mM zinc chloride, 1mM magnesium chloride, 0.05% sodium azide, 0.25% Tween-20, 0.2mg/mL ribonuclease, 4% hydroxypropyl-beta-cyclodextrin, 0.05% goat IgG, 0.0008% sulforhodamine B. The solution was decanted and the plate was washed twice with a solution containing: 128mM Tris pH7.2, 0.6M sodium chloride, 0.05% sodium azide, 0.24% Tween; then 250 μ L of a solution containing: 128mM Tris pH7.2, 0.6M sodium chloride, 0.05% sodium azide, 0.24% Tween. The plates were sealed with heat stable strips and incubated at 60 ℃ for 10 minutes. The solution was decanted and the plate was washed twice more with the following solutions: 128mM Tris pH7.2, 0.6M sodium chloride, 0.05% sodium azide. The plates were then washed twice with the following solutions: 40mM Tris pH 8.2, 100mM sodium chloride, 0.05% sodium azide. The solution was decanted and 100. mu.L of CDP-Star substrate was added to each well. The plates were incubated in the dark for 20 minutes and read on a DML luminometer. Table 15 shows the signal to noise ratios generated by the detection of 7 different HPV cloned targets at 100pg/mL by the method set forth above.

Table 15: typing of HPV16, 18, 31, 33, 35, 45 and 52 on 96-well plates

Example 13

Effect of Using a mask in combination with a sub-well plate

Alkaline phosphatase solution was added to CDP-Star substrate. This solution (20 μ L) was added to the indicated sub-wells on 384-well plates (Digene Corp.) and incubated for 20 minutes. The plates were then read on a 384 well plate luminometer at approximately 450nm with and without the addition of a mask (Digene Corp.). Table 16 shows that the S/N ratio is increased by more than 100-fold by the mask masking each well/sub-well. A sub-well plate with a mask has the advantage of overcoming the cross-talk between previously known sub-wells. Each cell of table 16 represents a single sub-well. Only two sub-wells have chemiluminescent substrates, which are indicated in bold.

Table 16: crosstalk between adjacent sub-apertures

Sub-wells with chemiluminescent substrate added are indicated in bold

Example 14

HPV typing in 384-sub-well plates

Detection of single and multiple types of HPV was performed in a sub-well plate format. In this example, a sample is added to each well and a single target migrates and hybridizes to the corresponding capture sequence probe. A detection reagent is added to the well where it binds to the target in the positive sub-well. Detection is carried out by chemiluminescence.

Type-specific capture oligonucleotides (two per HPV type) containing 5' -C12-amino linker were prepared at a final concentration of 5. mu.M (5 pmole/. mu.L) in 500mM disodium phosphate solution at pH 8.5 containing 1mM disodium EDTA. This solution was added at 20 μ L per well to each of the sub-wells of a sub-well plate (one type per sub-well), the plate was sealed with a heat stable strip and incubated at 42 ℃ for 4 hours. The solution was decanted and the wells were washed three times with TBS. The plates were then immediately used for HPV typing assays.

The plate was blocked with 6% casein in 100mM Tris-HCl, pH7.2, 0.05% sodium azide (1mL per well) for 30 min at room temperature. A standard sample (100pg/mL) consisting of dilutions of HPV plasmid (10kb) was prepared in deionized water consisting of: 100. mu.g/mL herring sperm DNA, 1M guanidine-HCl, 10mM Tris-HCl (pH 8.0), 10mM EDTA, 0.05% sodium azide. Samples (500. mu.l) were mixed with 250. mu.l of 1.75M NaOH in a microcentrifuge tube and incubated for 30 minutes at 70 ℃. Next, the following solutions of RNA probes (15 ng per experiment) were added to each tube: 0.125M sodium citrate, 0.125M sodium phosphate, 0.6M triethanolamine, 0.6N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, 0.83M glacial acetic acid, 3% polyacrylic acid, 5mM EDTA, 0.05% sodium azide, and 0.4% Tween-20(pH 3.6-4.1). The solution is now clear or yellowish. The contents of each tube (1mL) were transferred to the corresponding well on the sub-well plate. Incubate the plate at 65 ℃ for 1 hour with shaking at 1150 rpm. The plate was cooled to room temperature and the solution decanted. To each well was added an alkaline phosphatase solution (1mL per well) to which a mouse monoclonal anti-RNA DNA antibody was bound and a 30% blocking solution (100 mM Tris-HCl at pH7.2 containing 6% casein and 0.05% sodium azide), and the plate was incubated at 37 ℃ for 30 minutes, the alkaline phosphatase solution being: 100mM Tris-pH 7.4, 0.6M sodium chloride, 0.1mM zinc chloride, 1mM magnesium chloride, 0.05% sodium azide, 0.25% Tween-20, 0.2mg/mL ribonuclease, 4% hydroxypropyl-beta-cyclodextrin, 0.05% goat IgG, 0.0008% sulforhodamine B. The solution was decanted and the plate was washed twice with the following solutions: 128mM Tris pH7.2, 0.6M sodium chloride, 0.05% sodium azide, 0.24% Tween, then 1mL of the following solution was added to each well: 128mM Tris pH7.2, 0.6M sodium chloride, 0.05% sodium azide, 0.24% Tween. The plates were sealed with heat stable strips and incubated at 60 ℃ for 10 minutes. The solution was decanted and the plate was washed twice more with the following solutions: 128mM Tris pH7.2, 0.6M sodium chloride, 0.05% sodium azide, 0.24% Tween. The plates were then washed twice with 40mM Tris, 100mM sodium chloride, 0.05% sodium azide, pH 8.2. The solution was decanted and 20. mu.L of CDP-Star substrate (Applera Corp.) was added to each well. The plates were incubated in the dark for 20 minutes and read at approximately 450nm on a 384 luminometer with a mask as illustrated in example 13.

The results in table 17 indicate the specificity of detection of single and multiple HPV types in the sub-well plate. The signals shown in bold indicate the specificity of the detection. For example, the HPV16 target showed a positive signal when represented by the HPV16 probe alone, but did not show a signal with other HPV type probes. However, multiple infections show multiple signals.

Table 17: HPV typing in 384-sub-well plates using passive binding

Example 15

Comparison of HPV detection methods Using microplate and Microbeads

The two assay formats of the invention are compared, wherein the detection step is performed on a microplate (e.g., by chemiluminescent detection) or Luminex carboxylated microbeads (e.g., by fluorescent detection).

The target enrichment and target amplification steps were performed as set forth in example 6. The third step, which involves hybridization and detection, is performed in two ways: one was performed on Luminex carboxylated microbeads as set forth in example 6 and the other was performed on a microplate as set forth in example 14.

The results in Table 18 show that the plate assay can detect 500 input copies in 30 minutes of amplification with an S/N ratio of 9. At 1 hour of amplification, the sensitivity of the plate assay is at least an order of magnitude higher than the Luminex format.

Table 18: HPV detection using plate and bead detection format

Example 16

Effect of the proportion of nucleotides in random primers on isothermal amplification efficiency

Random primers were chemically synthesized using different combinations and ratios of nucleotides in the target amplification step of the method set forth in example 6. The results in Table 19 indicate that dG and T play a major role in achieving efficient amplification. Omission of dA had less pronounced effect and omission of dC had no effect. Isothermal amplification was performed for 2 hours using a pentamer primer. The entire experiment was performed as set forth in example 6. Relative efficiency was calculated as signal to noise ratio, where the efficiency of the standard primer composition was (1.0).

Table 19: the relative efficiency of target detection depends on the composition of the random primers

Example 17

HPV typing on microchip arrays

Detection of HPV types (both single and multiple types of HPV) can be performed on a microchip array format. Target nucleic acid capture and amplification were performed according to example 6. The formulation of reagents and washing protocols were carried out according to commercially available products and instructions for washing slides, membranes, chips, etc. According to example 6, the target detection step involves target denaturation and hybridization with an RNA probe or signal sequence probe.

In this example, hybridization of targets to sequence-specific capture sequence oligonucleotide probes was performed on an oligonucleotide dot array, rather than on microbeads, as illustrated in example 6. The term "spot array" refers to a two-dimensional series of elements on the surface of a solid support, where each element is an aliquot of an oligonucleotide deposited at a specific location. The solid support used in this manner comprises: glass slides, nylon membranes or microchips (glass or silicon). Additional examples And illustrations are found in "microarray And Cancer Research" ("microarray And Cancer Research") ed.j.warrington, Eaton Publishing, 2002.

Type-specific capture sequence oligonucleotide probes (two oligonucleotides per HPV type) modified with 5' -C12-amino linkers are spotted onto a solid support such as a microarray, each spot corresponding to a specific HPV type. The oligonucleotides are linked, for example, via primary amino groups. The solid support was equilibrated with 2 XSSC (sodium chloride and sodium citrate) buffer for 10 min at room temperature and then incubated in prehybridization buffer (2 XSSC/0.05% blocking reagent/5% dextran sulfate/0.1% SDS) containing 50. mu.g/mL denatured herring sperm DNA for 30 min at 37 ℃. Hybridization is then performed in the same prehybridization buffer to which the amplified target nucleic acid and RNA signal sequence probes are added. The target nucleic acid, RNA signal sequence probe, and type-specific capture sequence oligonucleotide probe in the hybridization solution hybridized when incubated for 3 hours at 37 ℃ with shaking.

Detection of the amplicon-containing spot can be performed in two ways: a) using a hybrid capture antibody (HC-Ab) labeled with Cy fluorescent dye, followed by scanning using a commercially available scanner; or b) use of alkaline phosphatase-labeled HC-Ab followed by the application of a substrate for precipitation (e.g., 5-bromo-4-chloro-3-indolyl-phosphate and tetrazolyl nitro blue: NBT/BCIP). In the latter case, the signal can be detected without instrumental assistance.

The foregoing description of various embodiments has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Obvious modifications or variations are possible in light of the above teachings. The embodiments discussed were chosen and described to provide illustration and its practical application to thereby enable one of ordinary skill in the art to utilize the various embodiments and with various modifications as are suited to the particular use contemplated. All such modifications and variations are intended to be included herein within the scope of this disclosure as determined by the appended claims when interpreted in accordance with the breadth to which they are fairly, legally, and equitably entitled.

Sequence listing

seqpctus0660603

SEQUENCE LISTING

<110>Nazarenko，Irina

Lorincz，Attila

Eder，Paul

Lowe，Brian

Mallonee，Richard

Thai，Ha

<120>DETECTION OF NUCLEIC ACIDS BY TARGET-SPECIFIC HYBRID CAPTURE

METHOD

<130>2629-4066

<140>TBD

<141>2005-11-07

<150>US 11/005,617

<151>2004-12-06

<150>US 10/971,251

<151>2004-10-20

<150>US 09/594,839

<151>2000-06-15

<160>129

<170>PatentIn version 3.3

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<211>32

<212>DNA

<213>HPV

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<211>33

<212>DNA

<213>HPV

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<212>DNA

<213>HPV

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<210>56

<211>34

<212>DNA

<213>HPV

<400>56

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<210>57

<211>42

<212>DNA

<213>HPV

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<210>58

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<212>DNA

<213>HPV

<400>58

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<210>59

<211>30

<212>DNA

<213>HPV

<400>59

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<210>60

<211>40

<212>DNA

<213>HPV

<400>60

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<210>61

<211>31

<212>DNA

<213>HPV

<400>61

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<210>62

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<212>DNA

<213>HPV

<400>62

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<210>63

<211>41

<212>DNA

<213>HPV

<400>63

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<210>64

<211>44

<212>DNA

<213>HPV

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<210>65

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<213>HPV

<400>65

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<210>66

<211>17

<212>DNA

<213>HPV

<400>66

ttttgtggtt ctgtgtg 17

<210>67

<211>14

<212>DNA

<213>HPV

<400>67

ttatgtggtt gcgc 14

<210>68

<211>15

<212>DNA

<213>HPV

<400>68

cgttgttgat cgtgc 15

<210>69

<211>15

<212>DNA

<213>HPV

<400>69

tgcaaacccg tgtag 15

<210>70

<211>16

<212>DNA

<213>HPV

<400>70

cagacaacga taaccg 16

<210>71

<211>15

<212>DNA

<213>HPV

<400>71

accaccacaa gcagc 15

<210>72

<211>17

<212>DNA

<213>HPV

<400>72

gtcctgttgt aaatgtg 17

<210>73

<211>15

<212>DNA

<213>HPV

<400>73

aggcgacact acgtc 15

<210>74

<211>15

<212>DNA

<213>HPV

<400>74

cgagctcccc tacaa 15

<210>75

<211>13

<212>DNA

<213>HPV

<400>75

ccccaccaag cga 13

<210>76

<211>15

<212>DNA

<213>HPV

<400>76

cctcacggga tactc 15

<210>77

<211>15

<212>DNA

<213>HPV

<400>77

tgcgacaact acagc 15

<210>78

<211>14

<212>DNA

<213>HPV

<400>78

ggattcgggc acta 14

<210>79

<211>18

<212>DNA

<213>HPV

<400>79

catataacac aggctcac 18

<210>80

<211>18

<212>DNA

<213>HPV

<400>80

tagtgctagg tgtagtgg 18

<210>81

<211>13

<212>DNA

<213>HPV

<400>81

acgcaggagg tgg 13

<210>82

<211>16

<212>DNA

<213>HPV

<400>82

tacatacacg cacgca 16

<210>83

<211>16

<212>DNA

<213>HPV

<400>83

ccacaaggca cattca 16

<210>84

<211>17

<212>DNA

<213>HPV

<400>84

gctacacgtg cacggcg 17

<210>85

<211>17

<212>DNA

<213>HPV

<400>85

gtgattaaat atggtgg 17

<210>86

<211>13

<212>DNA

<213>HPV

<400>86

gggacgttgg acg 13

<210>87

<211>14

<212>DNA

<213>HPV

<400>87

ggcaatgcgc taat 14

<210>88

<211>15

<212>DNA

<213>HPV

<400>88

caccgattct tccag 15

<210>89

<211>16

<212>DNA

<213>HPV

<400>89

cacctcacac aattcg 16

<210>90

<211>13

<212>DNA

<213>HPV

<400>90

ggatccggtg cag 13

<210>91

<211>19

<212>DNA

<213>HPV

<400>91

gtaaaagttg tgaacgatc 19

<210>92

<211>14

<212>DNA

<213>HPV

<400>92

ggtgctggag acaa 14

<210>93

<211>17

<212>DNA

<213>HPV

<400>93

acatctagag aacctag 17

<210>94

<211>15

<212>DNA

<213>HPV

<400>94

cacacatcag cgaca 15

<210>95

<211>16

<212>DNA

<213>HPV

<400>95

cagacaatac cgacag 16

<210>96

<211>13

<212>DNA

<213>HPV

<400>96

tgtgtggacc ggg 13

<210>97

<211>18

<212>DNA

<213>HPV

<400>97

ctgcaaatgt gtgtgtat 18

<210>98

<211>17

<212>DNA

<213>HPV

<400>98

gtcctctgca ctcacat 17

<210>99

<211>14

<212>DNA

<213>HPV

<400>99

acgtcctttg ccac 14

<210>100

<211>19

<212>DNA

<213>HPV

<400>100

ttctgagcaa ctatcatac 19

<210>101

<211>12

<212>DNA

<213>HPV

<400>101

ccctccgcaa cg 12

<210>102

<211>15

<212>DNA

<213>HPV

<400>102

ccccatcttg tttcc 15

<210>103

<211>16

<212>DNA

<213>HPV

<400>103

tcctacacgc ctagac 16

<210>104

<211>20

<212>DNA

<213>HPV

<400>104

tatagagaac tgctgtgttc 20

<210>105

<211>15

<212>DNA

<213>HPV

<400>105

gaaatagcgc cattg 15

<210>106

<211>14

<212>DNA

<213>HPV

<400>106

ctcaggggca acca 14

<210>107

<211>16

<212>DNA

<213>HPV

<400>107

atccataggg tccgac 16

<210>108

<211>11

<212>DNA

<213>HPV

<400>108

caatgcggcg c 11

<210>109

<211>16

<212>DNA

<213>HPV

<400>109

tataaacgag gtgcag 16

<210>110

<211>15

<212>DNA

<213>HPV

<400>110

gaaaatgccc tgcta 15

<210>111

<211>12

<212>DNA

<213>HPV

<400>111

acatgcgcca gg 12

<210>112

<211>19

<212>DNA

<213>HPV

<400>112

gagtaatgtg gtgtgtatg 19

<210>113

<211>15

<212>DNA

<213>HPV

<400>113

gcaaggcata ctgtg 15

<210>114

<211>15

<212>DNA

<213>HPV

<400>114

cactgacact tcgtg 15

<210>115

<211>18

<212>DNA

<213>HPV

<400>115

ggacaatcac cagtatta 18

<210>116

<211>18

<212>DNA

<213>HPV

<400>116

agtttgtgaa gtacatgg 18

<210>117

<211>12

<212>DNA

<213>HPV

<400>117

cgggtgatgg cc 12

<210>118

<211>18

<212>DNA

<213>HPV

<400>118

ccacttgtac tgtgtagg 18

<210>119

<211>18

<212>DNA

<213>HPV

<400>119

ctgtgtttaa ctatgggt 18

<210>120

<211>20

<212>DNA

<213>HPV

<400>120

tacaacagta tgtgtcagac 20

<210>121

<211>16

<212>DNA

<213>HPV

<400>121

aagacaggga gacagc 16

<210>122

<211>19

<212>DNA

<213>HPV

<400>122

cttataaaca atacacagg 19

<210>123

<211>20

<212>DNA

<213>HPV

<400>123

atgcactata gtaacacacc 20

<210>124

<211>19

<212>DNA

<213>HPV

<400>124

actccatttt agtgctgta 19

<210>125

<211>24

<212>DNA

<213>HPV

<400>125

tcaggaccca caggagcgac ccag 24

<210>126

<211>23

<212>DNA

<213>artificial

<220>

<223>Synthetic primer

<400>126

gcaccaaaag agaactgcaa tgt 23

<210>127

<211>24

<212>DNA

<213>artificial

<220>

<223>Synthetic primer

<400>127

catatacctc acgtcgcagt aact 24

<210>128

<211>20

<212>DNA

<213>HPV

<400>128

tattttatat gacacaatgt 20

<210>129

<211>30

<212>DNA

<213>HPV

<400>129

ggtttgtgct aacaataaat gtatccatag 30

Claims (24)

1. A method of detecting each of a plurality of target nucleic acids, comprising:

a) capturing the plurality of target nucleic acids onto a solid support by mixing the plurality of target nucleic acids, nucleic acid probes complementary to at least a portion of the target nucleic acids, and the solid support to form a plurality of enriched target nucleic acids, wherein one of the target nucleic acids and nucleic acid probes is RNA and the other is DNA;

b) amplifying the enriched nucleic acids or nucleic acid probes to form a plurality of amplified targets; and

c) detection of the plurality of amplified targets is performed by mixing the plurality of amplified targets, a selectable oligonucleotide that hybridizes to a first portion of the amplified targets, and a nucleic acid probe that is complementary to a second portion of the amplified targets to form a DNA: RNA hybrid oligonucleotide complex, wherein DNA: RNA hybrids are detected by a DNA: RNA hybrid specific binding agent and are selected by the oligonucleotide complex separation to detect each of the plurality of target nucleic acids.

2. The method of claim 1, wherein in the step of capturing, the solid support is bound to a DNA: RNA hybrid specific binding agent, thereby capturing the target nucleic acid onto the solid support.

3. The method of claim 1, wherein the DNA RNA hybrid-specific binding agent is selected from the group consisting of: RNA hybrid specific antibody, monoclonal antibody or polyclonal antibody or its fragment, protein, catalytic inactivation of ribonuclease H, and nucleic acid, nucleic acid aptamer or combined and formed three chain structure of oligonucleotide.

4. The method of claim 2, wherein the DNARNA hybrid-specific binding agent is a DNARNA hybrid-specific antibody.

5. The method of claim 2, wherein the DNA RNA hybrid-specific binding agent is a nucleic acid probe complementary to the target nucleic acid.

6. The method of claim 3, wherein the DNARNA hybrid-specific binding agent is detectably labeled.

7. The method of claim 6, wherein the label is detected by colorimetric, chemiluminescent, fluorescent, and light scattering methods.

8. The method of claim 1, wherein the captured target nucleic acid or nucleic acid probe is amplified by isothermal amplification.

9. The method of claim 8, wherein a DNA polymerase is used in the isothermal amplification.

10. The method of claim 8, wherein random primers are used in the isothermal amplification.

11. The method of claim 10, wherein the random primer is a pentamer.

12. The method of claim 10, wherein the random primers comprise a subset of disease-specific primer sequences.

13. The method of claim 1, wherein the selective oligonucleotide is bound to a solid support.

14. The method of claim 1, wherein the solid support is selected from the group consisting of: plates, microplates, slides, dishes, microbeads, particles, microparticles, cups, strips, chips, microchips, ribbons, membranes, microarrays, and test tubes.

15. The method of claim 14, wherein the solid support is fabricated from a material selected from the group consisting of: glass, silicon, plastic, polystyrene, polyethylene, polypropylene, and polycarbonate.

16. The method of claim 14, wherein the solid support is modified to comprise: the modification is performed so that the surface comprises carboxyl groups, amino groups, hydrazide, aldehyde groups, nucleic acid or nucleotide derivatives, primary amino groups and dyes.

17. The method of claim 1, further comprising adding a blocker probe.

18. A method of detecting each of a plurality of target nucleic acids, comprising:

a) hybridizing a plurality of target nucleic acids to nucleic acid probes complementary to at least a portion of the target nucleic acids to form DNA-RNA hybrids;

b) capturing said DNA RNA hybrid with a DNA RNA hybrid specific antibody bound to a solid support;

c) separating unbound target nucleic acid from the DNA-RNA hybrid;

d) amplifying the captured target nucleic acids or nucleic acid probes using random primers and a DNA polymerase to form a plurality of amplified targets;

e) hybridizing a nucleic acid probe complementary to a first portion of the amplified target to form a DNA-RNA hybrid;

f) hybridizing oligonucleotides bound to a plurality of solid supports to a second portion of the amplified target, wherein the solid supports are selective; and

g) selecting each of the plurality of amplified targets by the selective solid support; and

h) detecting each of the plurality of amplified targets by binding a DNA: RNA hybrid specific binding agent to the DNA: RNA hybrid, wherein the presence of a plurality of amplicons indicates the presence of the target nucleic acid.

19. The method of claim 18, wherein the solid support to which oligonucleotides are bound is a distinguishable microbead.

20. The method of claim 19, wherein the distinguishable beads are selected from the group consisting of detectable labels: fluorescent labels, gold labels and enzyme labels.

21. A multiplex method of detecting a plurality of target DNAs, comprising:

a) hybridizing a plurality of target DNAs with an RNA probe complementary to at least a portion of the target DNAs to form DNA-RNA hybrids;

b) capturing said DNA RNA hybrid with a DNA RNA hybrid specific antibody bound to the microbeads;

c) separating unbound nucleic acids from the DNA-RNA hybrids to form enriched targets;

d) amplifying the enriched target DNA using random primers and a DNA polymerase to form a plurality of amplified targets;

e) hybridizing an RNA probe complementary to a first portion of the amplified target DNA to form a DNA-RNA hybrid;

f) hybridizing a DNA oligonucleotide to a second portion of the target DNA, wherein the DNA oligonucleotide is bound to a plurality of selective microbeads; and

g) detecting the plurality of amplified target DNAs by binding the DNA: RNA hybrid-specific antibody to the DNA: RNA hybrid, and selecting each of the amplified targets according to a specific selective microbead.

22. The method of claim 1, wherein the nucleic acid probes and oligonucleotide sequences complementary to at least a portion of the target nucleic acid are selected from the group consisting of:

GTACAGATGGTACCGGGGTTGTAGAAGTATCTG [SEQ ID NO：1]；

CTGCAACAAGACATACATCGACCGGTCCACC [SEQ ID NO：2]；

GAAGTAGGTGAGGCTGCATGTGAAGTGGTAG [SEQ ID NO：3]；

CAGCTCTGTGCATAACTGTGGTAACTTTCTGGG [SEQ ID NO：4]；

GAGGTCTTCTCCAACATGCTATGCAACGTCCTG [SEQ ID NO：5]；

GTGTAGGTGCATGCTCTATAGGTACATCAGGCC [SEQ ID NO：6]；

CAATGCCGAGCTTAGTTCATGCAATTTCCGAGG [SEQ ID NO：7]；

GAAGTAGTAGTTGCAGACGCCCCTAAAGGTTGC [SEQ ID NO：8]；

GAACGCGATGGTACAGGCACTGCAGGGTCC [SEQ ID NO：9]；

GAACGCGATGGTACAGGCACTGCA [SEQ ID NO：10]；ACGCCCACCCAATGGAATGTACCC[SEQ ID NO：11]；TCTGCGTCGTTGGAGTCGTTCCTGTCGTGCTC [SEQ ID NO：12]；

*(TTATTATTA)CTACATACATTGCCGCCATGTTCGCCA [SEQ ID NO：13]；

(TTATTATTA)TGTTGCCCTCTGTGCCCCCGTTGTCTATAGCCTCCGT [SEQ ID NO：14]；

(TTATTATTA)GGAGCAGTGCCCAAAAGATTAAAGTTTGC [SEQ ID NO：15]；

(TTATTATTA)CACGGTGCTGGAATACGGTGAGGGGGTG [SEQ ID NO：16]；

(TTATTATTA)ACGCCCACCCAATGGAATGTACCC [SEQ ID NO：17]；

(TTATTATTA)ATAGTATTGTGGTGTGTTTCTCACAT [SEQ ID NO：18]；

(TTATTATTA)GTTGGAGTCGTTCCTGTCGTG [SEQ ID NO：19]；

(TTATTATTA)CGGAATTTCATTTTGGGGCTCT [SEQ ID NO：20]；

GCTCGAAGGTCGTCTGCTGAGCTTTCTACTACT [SEQ ID NO：21]；

GCGCCATCCTGTAATGCACTTTTCCACAAAGC [SEQ ID NO：22]；

TAGTGCTAGGTGTAGTGGACGCAGGAGGTGG [SEQ ID NO：23]；

GGTCACAACATGTATTACACTGCCCTCGGTAC [SEQ ID NO：24]；

CCTACGTCTGCGAAGTCTTTCTTGCCGTGCC [SEQ ID NO：25]；

CTGCATTGTCACTACTATCCCCACCACTACTTTG [SEQ ID NO：26]；

CCACAAGGCACATTCATACATACACGCACGCA [SEQ ID NO：27]；

GTTCTAAGGTCCTCTGCCGAGCTCTCTACTGTA [SEQ ID NO：28]；

(TTATTATTA)TGCGGTTTTGGGGGTCGACGTGGAGGC [SEQ ID NO：29]；

(TTATTATTA)AGACCTGCCCCCTAAGGGTACATAGCC [SEQ ID NO：30]；

(TTATTATTA)CAGCATTGCAGCCTTTTTGTTACTTGCTTGTAATAGCTCC [SEQ ID NO：31]；

(TTATTATTA)ATCCTGTAATGCACTTTTCCACAAA [SEQ ID NO：32]；

(TTATTATTA)GCCTGGTCACAACATGTATTAC [SEQ ID NO：33]；

(TTATTATTA)CAGGATCTAATTCATTCTGAGGTT [SEQ ID NO：34]；

TGCGGTTTTGGGGGTCGACGTGGAGGC [SEQ ID NO：35]；

GGCGCAACCACATAACACACAGAACCACAAAAC [SEQ ID NO：36]；

GTTCTACACGGGTTTGCAGCACGATCAACAACG [SEQ ID NO：37]；

CGCTGCTTGTGGTGGTCGGTTATCGTTGTCTG [SEQ ID NO：38]；

GACGTAGTGTCGCCTCACATTTACAACAGGAC [SEQ ID NO：39]；

CTCGCTTGGTGGGGTTGTAGGGGAGCTCGG [SEQ ID NO：40]；

GCTGTAGTTGTCGCAGAGTATCCCGTGAGG [SEQ ID NO：41]；

GTGAGCCTGTGTTATATGTAGTGCCCGAATCCC [SEQ ID NO：42]；

CCACCTCCTGCGTCCACTACACCTAGCACTA [SEQ ID NO：43]；

TGCGTGCGTGTATGTATGAATGTGCCTTGTGG [SEQ ID NO：44]；

AATTAGCGCATTGCCCCGTCCAACGTCCCG [SEQ ID NO：45]；

CGCCGTGCACGTGTAGCCACCATATTTAATCAC [SEQ ID NO：46]；

CGAATTGTGTGAGGTGCTGGAAGAATCGGTGC [SEQ ID NO：47]；

GATCGTTCACAACTTTTACCTGCACCGGATCC [SEQ ID NO：48]；

CTAGGTTCTCTAGATGTTTGTCTCCAGCACCCC [SEQ ID NO：49]；

CTGTCGGTATTGTCTGTGTCGCTGATGTGTG [SEQ ID NO：50]；

GATACACACACATTTGCAGCCCGGTCCACACA [SEQ ID NO：51]；

GGTGGCAAAGGACGTATGTGAGTGCAGAGGAC [SEQ ID NO：52]；

GCGTTGCGGAGGGGTATGATAGTTGCTCAGAAG [SEQ ID NO：53]；

GTCTAGGCGTGTAGGAGGAAACAAGATGGGG [SEQ ID NO：54]；

CTGAACACAGCAGTTCTCTATACCAATGGCGCTATTTC [SEQ ID NO：55]；

TTGGTTGCCCCTGAGCAGTCGGACCCTATGGATA [SEQ ID NO：56]；

GCGCCGCATTGCTGCACCTCGTTTATATAGCAGGGCATTTTC [SEQ ID NO：57]；

CCTGGCGCATGTCATACACACCACATTACTC [SEQ ID NO：58]；

CACGAAGTGTCAGTGCACAGTATGCCTTGC [SEQ ID NO：59]；

GCCATGTACTTCACAAACTGTTAATACTGGTGATTGTCCC [SEQ ID NO：60]；

CCTACACAGTACAAGTGGAGGCCATCACCCG [SEQ ID NO：61]；

GTCTGACACATACTGTTGTAACCCATAGTTAAACACAGG [SEQ ID NO：62]；

GCTGTCTCCCTGTCTTCCTGTGTATTGTTTAT AAGTGTATT [ SEQ ID NO: 63 ]; and

GTACAGCACTAAAATGGAGTTTGGTGTGTTACTATAGTGCATAC [SEQ ID NO：64]。

23. a composition for use in the method of claim 17, wherein the nucleic acid probe, oligonucleotide sequence and blocker probe complementary to at least a portion of the target nucleic acid are selected from the group consisting of:

GTACAGATGGTACCGGGGTTGTAGAAGTATCTG [SEQ ID NO：1]；

CTGCAACAAGACATACATCGACCGGTCCACC [SEQ ID NO：2]；

GAAGTAGGTGAGGCTGCATGTGAAGTGGTAG [SEQ ID NO：3]；

CAGCTCTGTGCATAACTGTGGTAACTTTCTGGG [SEQ ID NO：4]；

GAGGTCTTCTCCAACATGCTATGCAACGTCCTG [SEQ ID NO：5]；

GTGTAGGTGCATGCTCTATAGGTACATCAGGCC [SEQ ID NO：6]；

CAATGCCGAGCTTAGTTCATGCAATTTCCGAGG [SEQ ID NO：7]；

GAAGTAGTAGTTGCAGACGCCCCTAAAGGTTGC [SEQ ID NO：8]；

GAACGCGATGGTACAGGCACTGCAGGGTCC [SEQ ID NO：9]；

GAACGCGATGGTACAGGCACTGCA [SEQ ID NO：10]；ACGCCCACCCAATGGAATGTACCC[SEQ ID NO：11]；TCTGCGTCGTTGGAGTCGTTCCTGTCGTGCTC [SEQ ID NO：12]；

*(TTATTATTA)CTACATACATTGCCGCCATGTTCGCCA [SEQ ID NO：13]；

(TTATTATTA)TGTTGCCCTCTGTGCCCCCGTTGTCTATAGCCTCCGT [SEQ ID NO：14]；

(TTATTATTA)GGAGCAGTGCCCAAAAGATTAAAGTTTGC [SEQ ID NO：15]；

(TTATTATTA)CACGGTGCTGGAATACGGTGAG GGGGTG [SEQ ID NO：16]；

(TTATTATTA)ACGCCCACCCAATGGAATGTACCC [SEQ ID NO：17]；

(TTATTATTA)ATAGTATTGTGGTGTGTTTCTCACAT [SEQ ID NO：18]；

(TTATTATTA)GTTGGAGTCGTTCCTGTCGTG [SEQ ID NO：19]；

(TTATTATTA)CGGAATTTCATTTTGGGGCTCT [SEQ ID NO：20]；

GCTCGAAGGTCGTCTGCTGAGCTTTCTACTACT [SEQ ID NO：21]；

GCGCCATCCTGTAATGCACTTTTCCACAAAGC [SEQ ID NO：22]；

TAGTGCTAGGTGTAGTGGACGCAGGAGGTGG [SEQ ID NO：23]；

GGTCACAACATGTATTACACTGCCCTCGGTAC [SEQ ID NO：24]；

CCTACGTCTGCGAAGTCTTTCTTGCCGTGCC [SEQ ID NO：25]；

CTGCATTGTCACTACTATCCCCACCACTACTTTG [SEQ ID NO：26]；

CCACAAGGCACATTCATACATACACGCACGCA [SE QID NO：27]；

GTTCTAAGGTCCTCTGCCGAGCTCTCTACTGTA [SEQ ID NO：28]；

(TTATTATTA)TGCGGTTTTGGGGGTCGACGTGGAGGC [SEQ ID NO：29]；

(TTATTATTA)AGACCTGCCCCCTAAGGGTACATAGCC [SEQ ID NO：30]；

(TTATTATTA)CAGCATTGCAGCCTTTTTGTTACTTGCTTGTAATAGCTCC [SEQ ID NO：31]；

(TTATTATTA)ATCCTGTAATGCACTTTTCCACAAA [SEQ ID NO：32]；

(TTATTATTA)GCCTGGTCACAACATGTATTAC [SEQ ID NO：33]；

(TTATTATTA)CAGGATCTAATTCATTCTGAGGTT [SEQ ID NO.34]；

TGCGGTTTTGGGGGTCGACGTGGAGGC [SEQ ID NO：35]；

GGCGCAACCACATAACACACAGAACCACAAAAC [SEQ ID NO：36]；

GTTCTACACGGGTTTGCAGCACGATCAACAACG [SEQ ID NO：37]；

CGCTGCTTGTGGTGGTCGGTTATCGTTGTCTG [SEQ ID NO：38]；

GACGTAGTGTCGCCTCACATTTACAACAGGAC[SEQ ID NO：39]；

CTCGCTTGGTGGGGTTGTAGGGGAGCTCGG [SEQ ID NO：40]；

GCTGTAGTTGTCGCAGAGTATCCCGTGAGG [SEQ ID NO：41]；

GTGAGCCTGTGTTATATGTAGTGCCCGAATCCC [SEQ ID NO：42]；

CCACCTCCTGCGTCCACTACACCTAGCACTA [SEQ ID NO：43]；

TGCGTGCGTGTATGTATGAATGTGCCTTGTGG [SEQ ID NO：44]；

AATTAGCGCATTGCCCCGTCCAACGTCCCG [SEQ ID NO：45]；

CGCCGTGCACGTGTAGCCACCATATTTAATCAC [SEQ ID NO：46]；

CGAATTGTGTGAGGTGCTGGAAGAATCGGTGC [SEQ ID NO：47]；

GATCGTTCACAACTTTTACCTGCACCGGATCC [SEQ ID NO：48]；

CTAGGTTCTCTAGATGTTTGTCTCCAGCACCCC [SEQ ID NO：49]；

CTGTCGGTATTGTCTGTGTCGCTGATGTGTG [SEQ ID NO：50]；

GATACACACACATTTGCAGCCCGGTCCACACA [SEQ ID NO：51]；

GGTGGCAAAGGACGTATGTGAGTGCAGAGGAC [SEQ ID NO：52]；

GCGTTGCGGAGGGGTATGATAGTTGCTCAGAAG [SEQ ID NO：53]；

GTCTAGGCGTGTAGGAGGAAACAAGATGGGG [SEQ ID NO：54]；

CTGAACACAGCAGTTCTCTATACCAATGGCGCTAITTC [SEQ ID NO：55]；

TTGGTTGCCCCTGAGCAGTCGGACCCTATGGATA [SEQ ID NO：56]；

GCGCCGCATTGCTGCACCTCGTTTATATAGCAGGGCATTTTC [SEQ ID NO：57]；

CCTGGCGCATGTCATACACACCACATTACTC [SEQ ID NO：58]；

CACGAAGTGTCAGTGCACAGTATGCCTTGC [SEQ ID NO：59]；

GCCATGTACTTCACAAACTGTTAATACTGGTGATTGTCCC [SEQ ID NO：60]；

CCTACACAGTACAAGTGGAGGCCATCACCCG [SEQ ID NO：61]；

GTCTGACACATACTGTTGTAACCCATAGTTAAACACAGG [SEQ ID NO：62]；

GCTGTCTCCCTGTCTTCCTGTGTATTGTTTATAAGTGTATT [SEQ ID NO：63]；

GTACAGCACTAAAATGGAGTTTGGTGTGTTACTATAGTGCATAC [SEQ ID NO：64]；

and ACTCCAACGACGCAGA [ SEQ ID NO: 65 ];

TTTTGTGGTTCTGTGTG [SEQ ID NO：66]；

TTATGTGGTTGCGC [SEQ ID NO：67]；

CGTTGTTGATCGTGC [SEQ ID NO：68]；

TGCAAACCCGTGTAG [SEQ ID NO：69]；

CAGACAACGATAACCG [SEQ ID NO：70]；

ACCACCACAAGCAGC [SEQ ID NO：71]；

GTCCTGTTGTAAATGTG [SEQ ID NO：72]；

AGGCGACACTACGTC [SEQ ID NO：73]；

CGAGCTCCCCTACAA [SEQ ID NO：74]；

CCCCACCAAGCGA [SEQ ID NO：75]；

CCTCACGGGATACTC [SEQ ID NO：76]；

TGCGACAACTACAGC [SEQ ID NO：77]；

GGATTCGGGCACTA [SEQ ID NO：78]；

CATATAACACAGGCTCAC [SEQ ID NO：79]；

TAGTGCTAGGTGTAGTGG [SEQ ID NO：80]；

ACGCAGGAGGTGG[SEQ ID NO：81]；

TACATACACGCACGCA [SEQ ID NO：82]；

CCACAAGGCACATTCA [SEQ ID NO：83]；

GCTACACGTGCACGGCG [SEQ ID NO：84]；

GTGATTAAATATGGTGG [SEQ ID NO：85]；

GGGACGTTGGACG [SEQ ID NO：86]；

GGCAATGCGCTAAT [SEQ ID NO：87]；

CACCGATTCTTCCAG [SEQ ID NO：88]；

CACCTCACACAATTCG [SEQ ID NO：89]；

GGATCCGGTGCAG [SEQ ID NO：90]；

GTAAAAGTTGTGAACGATC [SEQ ID NO：91]；

GGTGCTGGAGACAA [SEQ ID NO：92]；

ACATCTAGAGAACCTAG [SEQ ID NO：93]；

CACACATCAGCGACA [SEQ ID NO：94]；

CAGACAATACCGACAG [SEQ ID NO：95]；

TGTGTGGACCGGG [SEQ ID NO：96]；

CTGCAAATGTGTGTGTAT [SEQ ID NO：97]；

GTCCTCTGCACTCACAT [SEQ ID NO：98]；

ACGTCCTTTGCCAC [SEQ ID NO：99]；

TTCTGAGCAACTATCATAC [SEQ ID NO：100]；

CCCTCCGCAACG [SEQ ID NO：101]；

CCCCATCTTGTTTCC [SEQ ID NO：102]；

TCCTACACGCCTAGAC[SEQ ID NO：103]；TATAGAGAACTGCTGTGTTC[SEQ ID NO：104]；

GAAATAGCGCCATTG [SEQ ID NO：105]；

CTCAGGGGCAACCA [SEQ ID NO：106]；

ATCCATAGGGTCCGAC [SEQ ID NO：107]；

CAATGCGGCGC [SEQ ID NO：108]；

TATAAAC GAGGTGCAG [SEQ ID NO：109]；

GAAAATGCCCTGCTA [SEQ ID NO：110]；

ACATGCGCCAGG[SEQ ID NO：111]；

GAGTAATGTGGTGTGTATG [SEQ ID NO：112]；

GCAAGGCATACTGTG [SEQ ID NO：113]；

CACTGACACTTCGTG [SEQ ID NO：114]；

GGACAATCACCAGTATTA[SEQ ID NO：115]；AGTTTGTGAAGTACATGG[SEQ ID NO：116]；

CGGGTGATGGCC [SEQ ID NO：117]；

CCACTTGTACTGTGTAGG [SEQ ID NO：118]；

CTGTGTTTA ACT ATGGGT [SEQ ID NO：119]；

TACAACAGTATGTGTCAGAC [SEQ ID NO：120]；

AAGACAGGGAGACAGC [ SEQ ID NO: 121 ]; CTTATAAACAATACACAGG [ SEQ ID NO: 122 ]; ATGCACTATAGTAACACACC [ SEQ ID NO: 123 ]; and ACTCCATTTTAGTGCTGTA [ SEQ ID NO: 124].

24. A composition comprising a sequence selected from the group consisting of SEQ ID NOs: 1-124.