CN101273143A - Multiplex polymerase chain reaction for genetic sequence analysis - Google Patents

Abstract

The present invention relates to a PCR method comprising: providing a biological sample suspected of containing one or more pathogenic nucleic acids; adding a plurality of PCR primers corresponding to genes found in the pathogen; and performing a polymerase chain reaction on the sample to A subpopulation of nucleic acid corresponding to the gene is amplified. The primers include at least one primer pair for each pathogen, and the primers contain a trailer sequence that is not homologous to any pathogen DNA or to any background DNA within the sample. The concentration of at least one primer within the polymerase chain reaction is no greater than about 100 nM.

Description

Multiplex polymerase chain reaction for genetic sequence analysis

This application is a reference to U.S. Provisional Patent Application No. 60/691,768 filed June 16, 2005; U.S. Provisional Patent Application No. 60/735,876 filed November 14, 2005; U.S. Provisional Patent Application No. filed November 14, 2005 60/735,824 and U.S. Provisional Patent Application No. 60/743,639, filed March 22, 2006, and U.S. Patent Application No. 11/422,425, filed June 06, 2006, and U.S. Patent Application No. 11/422,425, filed June 06, 2006 11/422,431 claims priority. This application is a continuation-in-part of US Patent Application Nos. 11/177,647, filed July 02, 2005; 11/177,646, filed July 02, 2005; and 11/268,373, filed November 07, 2005. These non-provisional applications are complementary to U.S. Provisional Patent Application No. 60/590,931, filed July 02, 2004; U.S. Provisional Patent Application No. 60/609,918, filed September 15, 2004; U.S. Provisional Patent Application No. 60/609,918, filed November 05, 2004 Application No. 60/626,500; US Provisional Patent Application No. 60/631,437, filed November 29, 2004, and US Provisional Patent Application No. 60/631,460, filed November 29, 2004 claim priority.

technical field

In general, the present invention relates to methods for the amplification of multiple genetic targets and the analysis of the amplification products using microarrays.

Background technique

Accurate and rapid identification of infectious pathogens causing acute respiratory infections (ARIs) in humans can be a key factor in the successful treatment of respiratory diseases, application of appropriate outbreak control measures, and effective use of precious antibiotics and antivirals. However, the clinical differential diagnosis of ARI is challenging due to the similarity of symptoms caused by different pathogens and the number and biodiversity of these agents. Currently, the most widely used methods for identifying respiratory pathogens are culture methods, immunoassays, and RT-PCR/PCR assays. Culture and immunoassay techniques are often specific for a particular pathogen and are thus limited to the detection of a single suspected agent at the species level and sometimes at the serotype level. In contrast, nucleic acid-based techniques such as RT-PCR/PCR are general, offering high sensitivity in the detection of all pathogens, including harsh or difficult-to-cultivate organisms. Due to the general nature of PCR, the technique can be applied to multiple agents simultaneously, increasing the chances of establishing a specific etiology (McDonough et al., "For the detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila and Bordetella pertussis in clinical samples Multiplex PCR for Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in clinical specimens (A mu1tiplex PCR for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Bordetella pertussis in clinical specimens)" Mol. Cell Probes, 19, 314-322 (2005)) and allows accurate detection Co-infection with one pathogen (Grondahl et al., "Rapid identification of nine organisms causing acute respiratory tract infections by single-tube multiplex reverse transcription PCR: a feasibility study (Rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-PCR: feasibility study)" J. Clin. Microbiol., 37, 1-7 (1999)).

Detection of several organisms by multiplexing within one reaction is required because ARI pathogens may be symptomatologically nonspecific. Therefore, assaying one pathogen at a time is inefficient and yields no information about possible co-infections. Several multiplex RT-PCR/PCR assays have been developed to address this issue (McDonough; Grondahl; Puppe et al., "Evaluation of a multiplex reverse transcriptase PCR ELISA for the detection of nine respiratory pathogens for the detection of nine respiratory tract pathogens)" J.Clin.Virol., 30, 165-174 (2004); Bellau-Pujol et al., "Development of a triplex RT-PCR assay for the detection of 12 respiratory RNA viruses (Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses)” J.Virol.Methods, 126, 53-63 (2005); Miyashita et al., “For Chlamydia pneumoniae, Mycoplasma pneumoniae and Multiplex PCR for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila incommunity-acquired pneumonia" Respir. Med., 98, 542-550 (2004); Osiowy, "By multiple inversion Direct detection of respiratory syncytial virus, parainfluenzavirus, and adenovirus in clinical respiratory specimens by a multiplex reversetranscription-PCR assay"J.Clin. Microbiol., 36, 3149-3154 (1998); Verstrepen et al., "Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with novel single -tube real-time reverse transcription-PCR assay)" J.Clin.Microbiol., 39, 4093-4096 (2001); Coiras et al., "Simultaneous detection of ten Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-PCRassays" J. Med. Virol., 72, 484-495 (2004); Coiras et al., "Using reverse line dot blot assay Oligonucleotide array for simultaneous detection of respiratory viruses using a reverse-line blot hybridization assay" J.Med.Virol., 76, 256-264 (2005); Gruteke et al. , "Practical implementation of a multiplex PCR for acute respiratory tract infections in children" J.Clin.Microbiol., 42, 5596-5603 (2004)), but the method Limited by the discriminatory power of current amplicon detection methods. Gel-based analytical methods are always limited to a limited class of pathogens whose products can be distinguished by size alone, while fluorescent reporter systems such as real-time PCR are limited by the number of fluorescent peaks that can be resolved with certainty—no more than 3 or 4. Therefore, there is a need for diagnostic assays that allow rapid differentiation and identification of pathogens such as ARI associated with disease syndromes with numerous potential etiologies.

Several techniques have been developed that allow simultaneous detection of more pathogens by RT-PCR/PCR methods. Multiplex identification of up to 22 respiratory pathogens has been performed with the MASSCODE ^™ multiplex RT-PCR system (Briese et al., "Diagnostic system for rapid and sensitive differential detection of pathogens" Emerg.Infect.Dis ., 11, 310-313 (2005)). Spotted (especially long oligonucleotide) microarrays have also been used with some success as a multiplex PCR analysis tool (Roth et al., "Oligonucleotides for the Laboratory Diagnosis of Bacteria Associated with Acute Upper Respiratory Tract Infection". Application of an oligonucleotide array for laboratory diagnosis of bacteria responsible for acute upper respiratory infections" J.Clin.Microbiol., 42, 4268-4274 (2004); Chizhikov et al., "Microarray Analysis of Microbial Virulence Factors (Microarray analysis of microbial virulence factors)” Appl.Environ.Microbiol., 67, 3258-3263 (2001); Chizhikov et al., “Detection and genotyping of human type A rotavirus by oligonucleotide microarray hybridization ( Detection and genotyping of human group Arotaviruses by oligonucleotide microarray hybridization)” J.Clin.Microbiol., 40, 2398-2407 (2002); Wang et al., “Microarray-based detection and genotyping of viral pathogens genotyping of viral pathogens)” Proc.Natl.Acad.Sci.USA, 99, 15687-15692(2002); Wang et al., “Viral discovery and sequence recovery using DNA microarrays” PLoS Biol., 1, E2 (2003); Wilson et al., "High-density microarray of small-subunit ribosomal DNA probes" Appl.Environ.Microbiol., 68, 2535-2541 (2002); Wilson et al., "Sequence-specific identification of 18 pathogenic microorganisms using microarray technology" Mol.Cell.Probes, 16, 119-127( 2002); Call et al., "Identifying antimicrobial resistance genes with DNA microarrays" Antimicrob.AgentsChemother., 47, 3290-3295 (2003); Call et al., "Mixed genome microarrays reveal Mixed-genome microarrays reveal multiple serotype and lineage-specific differences among strains of Listeria monocytogenes" J. Clin. Microbiol., 41, 632-639 (2003 ). A major limitation of these systems is the inability to distinguish closely related strains of the same organism, since the detected hybridization events may not be sensitive to local sequence diversity. For example, spotted microarray probes can non-specifically cross-hybridize to sequences that vary by as much as 25%—considering that if polymorphisms can be specifically defined, such elusive variant carriers can be highly distinguishable between strains. The fact that there is enough information about the system, this is an adverse event.

Identification at the strain level may be important in situations where closely related organisms may have disparate clinical outcomes and epidemiological patterns. In such cases, strains must be differentiated for proper handling and control. The clinically relevant Bordetella pertussis (Bordetella pertussis) and its clinically irrelevant sister species B. parapertussis provide a classic example. Another example is influenza viruses, for which distinction of vaccine-susceptible from vaccine-insensitive strains and circulating human isolates from possible zoonotic strains (eg avian H5N1) is of significant and obvious value.

Contents of the invention

The present invention includes a method comprising: providing a biological sample suspected of containing one or more pathogenic nucleic acids from within a predefined group of pathogens; adding a plurality of PCR primers corresponding to genes found within the predefined group of pathogens to within the sample; and performing a polymerase chain reaction on the sample to amplify a subpopulation of nucleic acid corresponding to the gene. The primers include at least one primer pair for each pathogen and the primers include a trailer sequence that is not homologous to any pathogen DNA within the sample or to any background DNA. The concentration of at least one primer within the polymerase chain reaction is not greater than about 100 nM.

Modes of Carrying Out the Invention

In the following description, which is intended to be illustrative rather than limiting, specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced in other embodiments that depart from these specific details. In other instances, detailed descriptions of well-known methods and apparatus are omitted so as not to obscure the description of the present invention with unnecessary detail.

Clinical syndromes are rarely specific to a single pathogen, so assays that allow the detection and differentiation of a large number of candidate pathogens will undoubtedly benefit public health efforts. The work presented here demonstrates the resequencing array (RA) approach combining multiplex RT-PCR/PCR, RA, and automated sequence similarity search and pathogen identification-RPM v.1 system for 20 species under broad-spectrum conditions. The clinical diagnosis and epidemiological monitoring capabilities of common respiratory pathogens and 6 biothreat agents. By combining the sensitivity of multiplex PCR amplification with RA specificity, the trade-off between specificity and sensitivity often encountered when evaluating diagnostic assays can be avoided. This was demonstrated using control samples spiked into healthy patient clinical samples either in extraction buffer, or as a complex mixture with extraction buffer. The data also show that using 101 throat swab samples from patients with influenza-like illness, the system provides equivalence with accepted RT-PCR/PCR-based and culture-based methods for both HAdV and influenza virus A. effect sensitivity.

Short oligonucleotide resequencing arrays (RA) can provide both species-level and strain-level identification of PCR amplicons from ARI pathogens. Strain-specific information, including unique polymorphisms from previously unknown variants, is provided by the ability of RAs tiled on the array to reveal sequence differences between hybridized target and prototype sequences (ProSeqs, see U.S. Patent Application No. 11/___,___ by Malanoski et al., entitled "Computer-Implemented Biological Sequence Identifier System and Method" and filed on the same date as the present application). Previous studies combined custom-designed respiratory pathogen microarrays (RPM v.1) with methods for microbial nucleic acid enrichment, random nucleic acid amplification, and automated sequence similarity Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays (Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays)” Genome Res., 16 (4), 527-535 (2006); Wang et al., "Rapid, Broad-spectrum Identification of Influenza Viruses by Resequencing Microarrays (Rapid, Broad-spectrum Identification of Influenza Viruses by Resequencing Microarrays)" Emerg.Infect.Dis., 12 (4), 638-646 (2006)). However, generic amplification methods have had limited success when processing clinical samples with lower pathogen titers. Disclosed herein is an improved multiplex PCR amplification strategy that alleviates sensitivity issues associated with random amplification of targets. Successful proof-of-concept experiments using clinical samples from patients exhibiting ARI demonstrated that high species-level concordance with standard reference assays (e.g. culture, College of American Pathologist [CAP ] authenticated PCR), while still yielding correct species-level and strain-level identifications by direct sequence reads within an improved assay time (8.5 hours). The results suggest that this approach is amenable to straightforward automation and miniaturization, and thus could lead to microarray-based platforms for diagnostic and monitoring purposes.

Polymerase chain reaction (PCR) is well known in the art. The methods of the invention use biological samples that may contain nucleic acids of a particular pathogen. The method does not require the presence of pathogenic nucleic acid and thus the method may be performed, for example, on specimens from healthy individuals. As an initial step, pathogenic nucleic acids can be extracted from clinical samples such as, but not limited to, nasal rinses, throat swabs, sputum, blood, or environmental samples. Clinical samples can be obtained from any biological species including, but not limited to, humans. Any type of pathogen can be detected, including but not limited to respiratory pathogens, enteric pathogens, and biological threats such as anthrax spores. Groups of pathogens can eg be defined at the species level or at the strain level.

The method involves PCR primers corresponding to genes that can be found in the pathogen. PCR primers are well known in the art. There is at least one primer pair for each pathogen. The primers used within the method have trailer sequences that are not homologous to the DNA of any pathogen or to any background DNA within the sample. The background DNA may be the DNA of the species from which the clinical sample was obtained. Potential trailer sequences can be generated randomly or additionally, and can be evaluated, for example, by comparison with genetic sequence databases such as GenBank. The trailer itself usually does not bind to pathogen DNA and can reduce the formation of primer dimers in PCR because the trailer is not complementary to any other primers. Suitable trailer sequences for use with specimens obtained from humans include, but are not limited to, CGATACGACGGGCGTACTACTAGCG (Primer L, SEQ ID NO. 1) and CGATACGACGGGCGTACTAGCGNNNNNNNNN (Primer LN, SEQ ID NO. 2).

A primer set within a single PCR may eg comprise at least 30, 40, 50, 60, 70, 80, 90 or 100 different primers. Primers corresponding to genes of different lengths can be used in the same PCR. For example, amplified nucleic acids less than 50, 100 or 200 nucleotides in length and greater than 3000 or 2000 nucleotides in length can be generated within a single PCR.

PCR using these primers can include other components or use equipment as generally known in the PCR art and as disclosed herein. Low concentrations of primers can be used within the reaction. One primer to all primers may be present at a concentration of no greater than about 100 nM. Lower concentrations such as 40-50 nM can be used.

A biological sample can be divided into multiple aliquots and an independent PCR using different primers performed on each aliquot. Aliquots can then be repooled after PCR. This can be done when using a large number of primers. The more primers used in a PCR, the higher the probability of primer-dimer formation. PCR can be better optimized with multiple aliquots with different primer mixes.

Following PCR, identification of the pathogen can be performed. This can be accomplished by contacting the sample with a microarray comprising a plurality of nucleic acid sequences that are at least partially complementary to the amplified nucleic acid, and allowing the amplified nucleic acid to hybridize to the complementary nucleic acid. Such methods are described in US Patent Application No. 11/177,646. Complementary nucleic acids can be, but are not limited to, 25 to 29 mers. The use of such short complementary nucleic acids reduces the likelihood of any mismatches hybridizing to the microarray. Complementary nucleic acids may include perfect match probes to at least one of the amplified nucleic acids and at most to all amplified nucleic acids and three different single nucleotide polymorphisms at the central position of each perfect match probe. This arrangement allows the determination of the complete sequence of the gene, which may allow the identification of the pathogen strain.

After hybridization, known methods such as fluorescence can be used to detect which complementary nucleic acids have hybridized to the amplified nucleic acid. Pathogens can then be identified based on which amplified nucleic acids are detected. This can be done with pattern recognition algorithms, where pathogens are identified based on which genes hybridize to the array. This can also be done based on genetic sequencing of hybrids as described above.

Molecular diagnostic techniques make it possible to quickly and sensitively identify etiological agents. Current methods such as PCR, RT-PCR, and spotted microarrays are prone to misidentification due to false positive and false negative test results, and are always adversely affected by the direct trade-off between sensitivity and specificity. The sample consists of a large number and variety of types of background organisms which may also contain similar regions to the target sequences used for diagnostic PCR amplification. The genetic complexity of non-target DNA, especially human DNA, can cause "false positive" product amplification due to cross-reactivity. In addition, viruses evolve at an extremely high rate through mutation and recombination events, leaving very sensitive assays in a state of frequent redesign or near-immediate obsolescence. Inherited variants are also clinically relevant because they may be associated with potentially significant antigenic variants for persistence of infection and response to therapy and/or vaccination. To study genetic variation with current PCR methods, an additional sequencing step is always required. The RPM v.1 method not only detects infectious agents at the species and strain levels, but also identifies subtle genomic differences without additional experiments. The method has also been demonstrated to be an efficient means for the simultaneous detection of up to seven pathogens with high sensitivity and specificity and allows unambiguous and reproducible sequence-based strain identification of pathogens in Moderate selection of prototype sequences on microarrays (ProSeqs). This can be used to enhance clinical management and epidemic outbreak response by allowing precise fingerprinting of each pathogen, antibiotic resistance distribution analysis, genetic drift/drift analysis, forensics and numerous other parameters. This capability could be invaluable for rapid detection of emergency diseases such as the avian influenza virus H5N1 and for bioterrorism events.

After describing the present invention, the following examples are given to illustrate specific applications of the present invention. These specific examples are not intended to limit the scope of the invention described within this application.

Example 1

RPM v.1 Chip Design - The RPM v.1 (Respiratory Pathogen Microarray) chip design includes 57 tiles that allow resequencing of 29.7kb sequences from 27 respiratory pathogens and biological warfare agents and was detailed in previous studies Described (Lin et al., "Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays" Genome Res., 16(4), 527-535(2006)). Briefly, RPM arrays consist of 25-mer perfectly matched probes representing sequences at each base within (and centered around) sequences selected from the genome of a target organism. In addition, for each perfectly matched probe, three mismatched probes representing the three possible single nucleotide polymorphisms (SNPs) at the central location were also tiled on the array. Thus, hybridization to a series of perfectly matched probes provides redundant presence/absence information, while hybridization to mismatched probes reveals strain-specific SNP data. On the chip, two pathogens, HAdV and influenza virus, were given more probe representation than the other pathogens. These probe representatives were selected based on clinical relevance to the population of immediate interest (US Army recruits in training). For HadV, partial sequences from ElA, hexon and fiber genes containing diagnostic regions for serotypes 4, 5 and 7 were collaged to detect all ARI-associated HadV. Similarly, the tiled regions for influenza virus A detection contain partial sequences from the hemagglutinin (subtypes H1, H3, and H5) gene, neuraminidase gene (subtypes N1 and N2), and matrix (matrix) gene . In addition to the 3 HAdVs and 3 influenza A viruses, the current RPM design allows the distinction between 15 other common respiratory pathogens known to cause ARI (i.e., "flu-like" symptoms early in infection) and 6 CDC category A viruses. Bioterrorism pathogens (Table 1). All control strains and field strains used to test the sensitivity and specificity of RPM v.1 and their sources are listed in Table 1.

Table 1: Analytical sensitivity of microarray-based assays against prototype control strains

Note: ^@ : plaque purified; ^* : minimum detection limit detected; ^# : target gene was constructed by BlueHeron Biotechnology (Bothell, WA) and cloned into pUC119.

Example 2

Clinical samples-archived throat swabs were collected from patients with symptoms of ARI at multiple Army recruit training centers, the US-Mexico border zone, and on board Navy ships deployed in 1999-2005. These throat swabs were immediately placed in chilled 2 mL vials containing 1.5 mL of viral transport medium (VTM), frozen and stored at -80°C or below to maintain virions during transport. Samples were then shipped to the Naval Health Research Center (NHRC, San Diego, CA), thawed, aliquoted, and tested for HAdV and influenza using a CAP-certified diagnostic RT-PCR/PCR and culture assay. to test. Frozen aliquots were subsequently submitted for microarray-based detection in blinded fashion.

Example 3

Nucleic acid extraction - use the MasterPure ^™ DNA Purification Kit (Epicentre Technologies, Madison, WI), omitting RNA digestion, or use the MagNA Pure Compact Nucleic Acid Isolation Kit I (MagNA Pure Compact Nucleic Acid Isolation Kit Kit I) (Roche Applied Science, Indianapolis, IN) was used to extract nucleic acids from clinical samples according to the manufacturer's recommended protocol.

Example 4

高产转录试剂盒(High Yield Transciption Kit)(艾姆拜恩(Ambion)，奥斯汀(Austin)，TX)从SP 6启动子进行体外转录。使用NAC1和TIM各60fg作为检查扩增效率和标本内抑制物存在的内对照。Internal Controls - Two Arabidopsis thaliana genes corresponding to NACl and triose phosphate isomerase (TIM) were selected as internal controls for reverse transcription (RT) and PCR reactions because they are unlikely to be naturally present in clinical samples Inside. The two plasmids pSP64poly(A)-NAC1 and pSP64poly(A)-TIM containing about 500bp of these two genes were obtained by Dr.Norman H from The Institute for Genome Research (Rockville, MD). . Kindly provided by Lee. NAC1 was amplified by PCR with SP6 and M13R primers, and the PCR product was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA). To generate RNA from pSP64poly(A)-TIM, this plasmid was linearized with EcoRI and used

The High Yield Transcription Kit (Ambion, Austin, TX) performs in vitro transcription from the SP 6 promoter. Use 60 fg each of NAC1 and TIM as an internal control for checking the amplification efficiency and the presence of inhibitors in the specimen.

Example 5

Primer Design and Multiplex RT-PCR Amplification - Assigning primers to two separate reactions simplifies primer design and optimization. Fine-tuning of both mixes (replacing primers that amplified poorly with the new mix) was performed to ensure adequate amplification from the 26 targeted pathogens (West Nile virus was included on the array but not included in the amplification protocol) would be adequately amplified. ) to allow hybridization. Gene-specific primer pairs (listed in Tables 2(a) and 2(b)) for all targets on the RPMv.1 chip were designed to ensure excellent amplification efficiency for multiplex PCR. All primers were designed to have similar annealing temperatures, and known sequences were checked with the BLAST program for uniqueness using a full search of the GenBank database. All primers were checked for potential hybridization with other primers to reduce the possibility of primer-dimer formation. In addition, we adapted the method developed by Shuber et al., "A simplified procedure for developing multiplex PCRs" Genome Res., 5, 488-493 (1995) and Brownie et al., "Reducing primer dimerization in PCR." A method developed by "The elimination of primer-dimeraccumulation in PCR" Nucleic Acids Res., 25, 3235-3241 (1997), aimed at adding a 22bp linker sequence (primer L) to the 5' primer used in this study end to further inhibit primer-dimer formation. The reverse transcription (RT) reaction was carried out in a 20 μl volume containing 500 μM, 40 U of each of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , dATP, dCTP, dGTP, and dTTP RNase OU ^™ , 10 mM DTT, 2 μM Primer LN, 200 U Superscript III (Invitrogen Life Technologies, Carlsbad, CA), 60 fg each of the two internal controls (NAC1 and TIM) and 5-8μl extracted clinical specimens or laboratory controls. Performed within a Peltier Thermal Cycler-PTC240 DNA Engine Tetrad 2 (MJ Research Inc., Reno, NV) using the manufacturer's recommended protocol reaction.

Table 2(a): List of PCR primers within primer mix A for multiplex PCR

Table 2(b): List of PCR primers within primer mix B for multiplex PCR

^* RSV: respiratory syncytial virus

The RT reaction product was divided into two 10 μl volumes for two different multiplex PCR reactions. Primer mix A contained 19 primer pairs and amplified 18 gene targets from 3 different influenza A viruses, 1 influenza virus B, 3 serotypes of HadV and an internal control (TIM). Primer mix B contained 38 primer pairs and amplified the remaining 37 gene targets and another internal control (NAC1). PCR reactions were carried out in a volume of 50 _μl containing 400 μM, 1 U of each heat-susceptible urine Pyrimidine-DNA Glycosylase (USB, Carlsbad, CA), 2 μM Primer L, 40 nM each primer from mix A or 50 nM each primer from mix B, 10 U Platinum (Platinum) Taq DNA polymerase (Invitrogen Life Technologies , Carlsbad, CA) and 10 μl of RT product. The amplification reaction was carried out in a Peltier Thermal Cycler-PTC240 DNA Engine Tetrad 2 (MJ Research Inc., Reno, NV), with initial incubation at 25°C for 10 minutes, initial denaturation at 94°C for 3 minutes, followed by 5 cycles: 94°C 30 s hold, 90 s at 50°C, 120 s at 72°C; followed by 35 cycles of 30 s at 94°C, 120 s at 64°C and a final 5 min extension at 72°C. Amplified products from the two PCR reactions were pooled into one volume and subjected to purification and processing prior to hybridization to the RPM v.1 chip (see below).

Example 6

Microarray Hybridization and Processing - The two PCR product mixtures are combined after amplification for fragmentation and hybridization to the microarray. Microarray hybridization and processing were performed according to the manufacturer's recommended protocol (Affymetrix Inc., Santa Clara, CA) with the following modifications. Purified PCR products were fragmented at 37°C for 5 minutes and subsequently labeled with biotin-N6-ddATP for 30 minutes at 37°C. Hybridization was carried out in a rotary electric oven at 45°C and 60 rpm for 2 hours. After scanning, GCOS software was used to downscale the original image (.DAT) file to a simplified file format (.CEL file) while assigning densities to each corresponding probe location. Finally, use the GDAS software to apply the built-in version of ABACUS (Cutler et al., "High-throughput variation detection and genotyping using microarrays" Genome Res., 11, 1913-1925 (2001) ) algorithm in order to generate correct base call (base call) estimates, comparing the respective strengths of the sense probe set and the antisense probe set. To increase the percentage of base calls, parameters were adjusted to allow the maximum allowable base calls (see below). Sequences from the base calls generated for each tile of the resequencing array method are then exported from within GDAS as FASTA format files.

- filter conditions

No signal threshold = 0.500 (default = 1.000000)

Weak signal doubling threshold = 20000.000 (default = 20.000000)

Huge signal-to-noise ratio (SNR) threshold = 20.000000 (default = 20.000000)

- Algorithm parameters

Chain quality threshold = 0.000 (default = 0.000000)

Overall quality threshold = 25.0000 (default = 75.000000)

Maximum fraction of heterozygous responses = 0.99000 (default = 0.900000)

Pattern type (0=heterozygote, 1=homozygote)=0

Full Response Quality Threshold = 0.500 (default = 2.000000)

- Terminal reliability principle

· Minimum fraction of responses within adjacent probes = 1.0000 (cannot be filtered)

• Response min fraction of samples = 1.0000 (cannot be filtered)

Example 7

Automated Pathogen Identification Algorithm (NA Sequence-Based Pathogen Identification) - The raw output sequences generated from microarray hybridizations and scans are processed using an algorithm that uses sequence similarity comparisons to database records and Identify pathogens. A new software program , Computer -Implemented Biological Sequence -based I dentifier system version 2 (CIBSI 2.0), was developed to identify pathogens by incorporating previous research in resequencing ( REPI) program (Lin et al., "Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays" Genome Res., 16(4), 527-535(2006)) tasks and, in addition, thoroughly analyze the results by making decisions that were previously made manually. A more extensive discussion of this approach, including the modified REPI algorithm, is described in detail in US Patent Application No. 11/422,431, filed June 6, 2006.

Example 8

Quantification of pathogens - The specificity of the assay was demonstrated using a variety of prototype strains and clinical samples. The results showed no discernible interference between targets. The analytical sensitivity of the RPM v.1 assay was subsequently assessed using serial ten-fold dilutions of the nucleic acid template of the prototype strain. Table 1 shows the lowest detectable dilution of pathogen for each pathogen. The results show that the sensitivity range for the prototype strain is ^10-103 genome copies per reaction, which is comparable to that of the standard multiplex RT-PCR/PCR method. It should be noted that genome copy number should not be equated with viability counts (plaque forming units) because for respiratory pathogens genome copy number is usually at least an order of magnitude higher than viability counts if not several orders of magnitude. The ability of RPM v.1 to identify and distinguish between close genetic neighbors was first demonstrated with a more specific protocol and has subsequently been reproduced with this protocol. Sequences generated from 17 different serotypes of human adenovirus (HAdV) revealed that the assay could distinguish multiple ARI-associated HAdV strains and demonstrated that the assay could be used to detect a broad class of variants (Table 3). Cross-hybridization of targets was only observed on the HAdV hexon gene between different serotypes; however this did not affect the positive identification of the correct targeted pathogen.

Table 3: Differentiation of multiple FRI-causing HAdVs using RPM v.1

For sensitivity assessment, real-time PCR assays were performed on an iCycler or MYIQ ^™ instrument (Bio-Rad Laboratories, Hercules, CA) to determine the number of adenoviral genomes in each sample. The results of the samples were compared by using the fiber-specific primers Ad4F-F and Ad4F-R (Table 4) with 10-fold serial dilutions of the HAdV-4 prototype genomic DNA template of known copy number ( ¹⁰¹ to ¹⁰⁶ copies). The results were compared. HAdV-4 genome copy number was calculated by measuring the DNA concentration from purified viral DNA and using the following conversion factor: 0.384fg = about 35kb (-35kb) of a single adenovirus genome. Real-time PCR reactions were performed in a 25 μl reaction volume containing 2.5 μl FastStart reaction mix SYBR Green I (Roche Applied Science, Indianapolis, IN), 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM 200 μM each of MgCl ₂ , dATP, dTTP, dGTP, dCTP, 200 nM primers and adenoviral genomic DNA (1-4 μl clinical specimen or DNA extract). Amplification reactions were performed with initial denaturation at 94°C for 10 minutes, followed by 40 cycles of 94°C for 20 seconds and 60°C for 30 seconds.

A similar assay was performed to distinguish influenza A virus by using specific primers (Table 4) and RT-PCR/PCR conditions as previously described (Stone et al., "Rapid detection and simultaneous subtype differentiation by real-time PCR subtype differentiation of influenza Aviruses by real time PCR)” J.Virol.Methods, 117, 103-112(2004); Hardegger et al., “Rapid detection of Mycoplasma pneumoniae in clinical samples by real-time PCR real-time PCR)” J. Microbiol. Methods, 41, 45-51 (2000); Corless et al., “Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae using real-time PCR in cases of suspected meningitis and sepsis and Streptococcus pneumoniae (Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcuspneumoniae in suspected cases of meningitis and septicemia using real-time PCR)" J.Clin.Microbiol., 39, 1553-1558 (2001); etc., "Direct and rapid identification and genogrouping of meningococci and porA amplification by LightCycler PCR" J.Clin.Microbiol., 40, 4531-4535 ( 2002); Vabret et al., "Direct diagnosis of human respiratory viruses 229E and OC43 by the polymerase chain reaction" J.Virol.Methods, 97, 59-66 (2001)) to determine the genome copy number of other pathogens.

Table 4. List of PCR primers used for quantitative real-time PCR

双标记探针在5’端含有6-羧基-荧光素(FAM)作为荧光报告染料并在3’端含有Black Hole猝灭剂。 ^* RSV: respiratory syncytial virus,

The dual-labeled probe contains 6-carboxy-fluorescein (FAM) as a fluorescent reporter dye at the 5' end and a Black Hole quencher at the 3' end.

references:

Stone et al., "Rapid detection and simultaneous subtype differentiation of influenza A viruses by realtime PCR" J.Virol.Methods, 117, 103-112 (2004).

Vabret et al., "Direct diagnosis of human respiratory coronaviruses 229E and OC43 by the polymerase chain reaction" J.Virol.Methods, 97, 59-66 (2001)

Hardegger et al., "Rapid detection of Mycoplasma pneumoniae in clinical samples by real-time PCR" J. Microbiol. Methods, 41, 45-51 (2000)

Corless et al, "Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR)" J. Clin. Microbiol., 39, 1553-1558 (2001)

Mentel et al., "Real-time PCR to improve the diagnosis of respiratory syncytial virus infection" J.Med.Microbiol., 52, 893-896 (2003)

等，“通过LightCycler PCR直接并快速对脑膜炎球菌及porA扩增鉴定和基因分型(Direct and rapid identification and genogrouping ofmeningococci and porA amplification by LightCycler PCR)”J.Clin.Microbiol.，40，4531-4535(2002)

etc., "Direct and rapid identification and genogrouping of meningococci and porA amplification by LightCycler PCR" J.Clin.Microbiol., 40, 4531-4535 (2002)

Example 9

Simultaneous Detection and Differentiation of Respiratory Pathogens - Previous studies have demonstrated that one of the outstanding benefits of using the RPM v.1 assay for pathogen detection, in addition to the precise identification of individual pathogen species, is the ability to detect co-infections. In this study, the ability of the RPM v.1 assay to simultaneously identify multiple pathogens was further evaluated by preparing various combinations of pathogen templates (Table 5 and Table 6). Serial dilutions of template were used to estimate detection sensitivity and specificity for multiple pathogens. Nucleic acid templates containing 10 ⁶ -10 ³ genome copies per reaction of each pathogen, HAdV-4, S. pyogenes, M. pneumoniae and Y. pestis, were pooled and tested with the RPM v.1 array. These results demonstrate that the assay allows reproducible sequence-based identification of all 4 pathogens even at a minimum concentration of ¹⁰³ genome copies per target per reaction (Table 5). The fact that there were no discernible interferences in this complex mixture further supports the nucleotide base calling capability of RA and the robustness of the accompanying identification algorithm, even in complex mixtures.

To further estimate the efficiency of this method for detecting multiple pathogens in complex ^{mixtures, 3-7 cultured organisms were cultured at various titers [102-105 (} colony-forming units or plaque-forming units)/mL] Added to pooled nasal rinses collected from volunteers and 150 μl of prepared samples were used for testing. Preliminary results reveal that the method allows unambiguous simultaneous detection of 7 pathogens at a minimum titer of 100 colony forming units (plaque forming units)/mL: HAdV-4, HAdV-7, Bacillus anthracis, Influenza virus A - H1N1, Parainfluenza virus 1, RSV-A, Mycoplasma pneumoniae and Streptococcus pyogenes (Table 6). Further evaluation with different combinations of 7 pathogens showed that the RPM v.1 assay could detect 6 of them simultaneously. Among these pathogens, HAdV-4, Bacillus anthracis, influenza virus A-H1N1, RSV-A, and Mycoplasma pneumoniae were detected at a minimum titer of 100 colony-forming units (plaque-forming units)/mL, while chain pyogenes Cocci were detected at 1000 cfu/mL (Table 6). Yersinia pestis could not be detected even at the highest concentrations. This was attributed to the inadequacy of the nucleic acid extraction protocol used for the intact Y. pestis pathogen, as 1000 copies of the genome of Y. pestis could be detected when using the purified nucleic acid template (Table 1 and table 5). For further confirmation, RPMv.1 was tested as cultured organisms on the same combination of 4 pathogens detected using purified nucleic acid templates (Table 5). The results showed that the assay could reproducibly detect HAdV-4 and Mycoplasma pneumoniae at 100 colony-forming units (plaque-forming units)/mL without detection failure, and was less sensitive to Streptococcus pyogenes (1000 colonies forming units/mL), but Yersinia pestis could not be detected. When testing for 3 pathogens simultaneously, in this example Bacillus anthracis, influenza virus A-H1N1, and HCoV-229E or RSV-A, the assay detected titers as low as 100 colony-forming units (plaque-forming units) /mL of all 3 pathogens (Table 6). These results suggest that the RA-based method is an effective means for the direct detection and typing of multiple pathogens from nasal rinse samples, with the benefit of high sensitivity and specificity for detection of co-infection with at least 7 pathogens. The method will be used in routine diagnostic and epidemiological investigations of these pathogens within populations, providing new information on the incidence of multiple pathogens.

Table 5: Simultaneous detection of multiple nucleic acid templates by RPM v.1

NOTE: Samples were generated by mixing purified nucleic acid templates in TE buffer to yield ¹⁰⁶ genome copies/ul stock. Starting from this concentration, 10-fold serial dilutions in TE buffer were prepared.

Table 6; Simultaneous detection of multiple pathogens by RPM v.1

NOTE: Samples were generated by mixing culture samples with pooled nasal rinses collected from normal volunteers to yield ¹⁰⁵ colony forming units (plaque forming units)/ml. Starting from this concentration, 10-fold serial dilutions containing pooled nasal rinses collected from normal volunteers were prepared. For each dilution, 150 μl of sample was used for the RPM v.1 method. BA-Bacillus anthracis (Sterne), H1N1-Influenza virus A-H1N1, HCoV229E-human coronavirus 229E, HAdV-human adenovirus, GAS-streptococcus pyogenes (group A streptococcus), MP-mycoplasma pneumoniae, PIV1: Parainfluenza virus 1, RSV-respiratory syncytial virus, YP-Yersinia pestis. +: detected, -: not detected.

Example 10

Evaluation of Clinical Specimens - Following successful demonstration of the capability of the RPM v.1 assay for pathogen detection, the assay was used for the prospective diagnosis and retrospective diagnosis of infections causing ARI. Clinical specimens collected primarily from military recruits presenting with ARI were used to compare the use of microarray-based diagnostics with more established methods for the detection of respiratory pathogens. Samples (n=101) consisted of throat swabs with clinically documented respiratory disease in viral transport medium. Samples were randomly selected from within groups that had tested positive for HAdV or influenza virus at the NHRC using CAP-approved diagnostic methods (cell culture and/or PCR). These samples were double-blinded (randomly renumbered and separated from relevant clinical records) and sent to the Naval Research Laboratory (NRL) for RPM v.1 testing. Comparative experiments were performed by two independent laboratories and sample identities were only revealed after the results had been finalized. For influenza virus A, the RPMv.1 method showed a detection sensitivity of 87% and a specificity of 96% and an overall agreement of 92% relative to the initial diagnosis (Table 7). For adenovirus, the sensitivity of RPM v.1 detection was 87%, the specificity was 97%, and the overall coincidence was 97% (Table 7). When the RPM v.1 results were further compared with those of the culture and PCR methods, the data showed comparable detection sensitivity and specificity to either the culture assay or the PCR assay (Table 8). The data indicate that RPM v.1 has better sensitivity and specificity than culture and PCR, which is expected since molecular methods are generally more sensitive than culture, and the sequencing power of RPM v.1 provides greater sensitivity than PCR. Higher specificity (Table 9). The data also strongly demonstrate the ability of microarray-based diagnostics to correctly identify clinically relevant influenza A strains in uncultured patient specimens.

Table 7: Evaluation of RPM v.1 for the detection of Adenovirus, Influenza A and Negative Controls in Clinical Samples

认证的PCR；^*将CAP认证的PCR阴性样品中的一个样品对流感病毒A进行培养，RPM v.1显示同时感染HAdV-4和流感病毒A，证实另一个阴性样品具有低滴度的HAdV-4；^￠3份流感病毒A培养法阳性样品也不能通过定量实时PCR得以检测，表明模板降解。

Authenticated PCR; ^* One of the CAP-authenticated PCR-negative samples was cultured for Influenza A, RPM v.1 showing co-infection with HAdV-4 and Influenza A, confirming that the other negative sample had low titers of HAdV- 4; ^￠ 3 samples positive for influenza virus A culture method could not be detected by quantitative real-time PCR, indicating template degradation.

Table 8: Comparison of RPM v.1 to culture and real-time PCR assays for detection of influenza virus A positive and negative controls in 40 clinical samples

Table 9: Comparison of culture method and real-time PCR identification method for influenza virus A positive control and negative control detection in 40 clinical samples

This study demonstrates the ability of this assay to identify influenza virus subtypes and track genetic changes. This is especially important for influenza virus epidemiology, as antigenic drift is a mechanism by which influenza viruses escape immune pressure induced by prior natural exposure and vaccination. Analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences generated from RPM v.1 reproduced known lineage changes through antigenic drift from 1999-2005 (Table 10). Seven influenza A/H3N2 clinical specimens collected before the 2003-2004 influenza season were identified as belonging to the A/Panama/2007/99-like lineage, while nine influenza A/H3N2 samples collected during the 2003-2004 influenza season Unmistakably carry characteristic A/Fujian/411/2002-like lineage nucleotide substitutions within the HA gene. A shift from the A/Fujian/411/2002 strain to the A/California/7/2004 strain was evident in 18 influenza A/H3N2 samples collected during the 2004-2005 influenza season. Three samples were identified as A/Fujian/411/2002-like strains, while the remaining samples showed characteristic California-like nucleotide substitutions within the HA gene. 2 samples collected during the same time period could only be identified as influenza virus A/H3N2. This is due to poor amplification and/or hybridization of the target, resulting in insufficient information for strain-level identification. Two samples of influenza virus A/H1N1 collected in 2000-2001 were identified as being closely related to A/New Caledonia/20/99.

Table 10: Influenza strains and lineage identification using RPM v.1

Note: ^* indicates that only a single plant has been identified

In addition to detecting single pathogens in clinical samples, multiple co-infections such as HAdV-4/influenza virus A, HAdV-4/S. pyogenes, and influenza A/M. show). These co-infections were performed using published type-specific PCR assays (Stone et al., "Rapid detection and simultaneous subtype differentiation of influenza A viruses by real time PCR" J. Virol. Methods, 117, 103-112 (2004); Hardegger et al., "Rapid detection of Mycoplasma pneumoniae in clinical samples by real-time PCR" J.Microbiol.Methods, 41, 45 -5 (2000)) and in-house specific PCR primers (Table 4) (data not shown) for further validation. In addition, the assay detected S. pneumoniae in 26% of clinical samples and N. meningitidis in 16% of clinical samples. The presence of S. pneumoniae and N. meningitidis was verified within a subset of 40 clinical samples (limited by the available volume of samples) by a published species-specific quantitative real-time PCR assay (data not shown) . Streptococcus pneumoniae and N. meningitidis are well known commensal bacteria in the mouth and upper respiratory system, so it is generally not surprising to find these bacteria in clinical samples. However, quantitative real-time PCR data showed that despite low titers (≤10 ³ genome copies/μl) of S. pneumoniae and N. meningitidis present in most clinical samples, 32% of influenza-positive samples carried high titers pneumoniae (7/25) or N. meningitidis (1/25) (≥10 ⁵ genome copies/μl) (data not shown). The high titers of bacteria present in these clinical samples may be due to virus-induced bacterial superinfection.

Example 11

Multiplex PCR protocol with primers L and LN - The following is a detailed protocol of the example method.

Preparation

SP6试剂盒1. Preparation of TIM RNA from pSP64poly(A)-TIM-

SP6 Kit

(Ambion, catalog number 1330)

a) 1 μg of pSP64poly(A)-TIM linearized with EcoRI enzyme

xμl pSP64poly(A)-TIM

2 μl 10× EcoRI buffer

2 μl EcoRI (NEB, catalog number R0101S)

(16-x)μl H ₂ O

         

20μl total volume

The reactions were incubated at 37°C for 5 hours.

b) Terminate the restriction digest by adding:

1/20 volume 0.5M EDTA (1μl)

1/10 volume of 3M sodium acetate (2μl)

2 volumes of ethanol (40 μl)

c) Mix well and freeze at -20°C for 20 minutes. The DNA was then pelleted in a microcentrifuge at top speed for 15 minutes.

d) Remove the supernatant, re-spin the tube for a few seconds, and remove residual liquid with a very sharp tip. Resuspend in 20 μl nuclease-free water.

e) Place the RNA polymerase enzyme mix on ice

f) Vortex to mix 10× reaction buffer with 4 ribonucleotide solutions

g) (ATP, CTP, GTP and UTP) until they are completely dissolved in solution.

h) Ribonucleotides were stored on ice once thawed, but the 10X reaction buffer was maintained at room temperature during assembling reactions.

i) All reagents should be microcentrifuged briefly prior to opening to prevent loss and/or contamination of material that may be present around the edge of the tube.

j) Form the following transcription reaction at room temperature

16 μl linearized pSP64poly(A)-TIM

16μl NTP (ATP, GTP, CTP, UTP each 4μl)

4 μl 10× reaction buffer

4 μl enzyme mix

           

40μl total volume

The reactions were incubated at 37°C for 6 hours.

k) Probe Quant ^TM G-50 microcolumn (Amersham, Cat. No. 27533501) for purification of RNA products

1) Measure the O.D. of recovered RNA and dilute to create a 60 fg/μl stock solution.

2. Prepare NAC1 DNA fragments from pSP64poly(A)-NAC1-PCR with platinum Taq DNA polymerase (Invitrogen, catalog number 10966-034)

1μl pSP64poly(A)-NAC1 (1ng/μl)

5 μl 10×PCR buffer

2μl 50mM _MgCl2

1 μl 10mM dNTP mix

1μl SP6 (10μM)

5’-ATT TAG GTG ACA CTA TAG AAT-3’

1μl SP6 (10μM)

5’-CAG GAA ACA GCT ATG ACC ATG-3’

0.5 μl Platinum Taq polymerase

38.5 μl _H2O

           

50μl total volume

Run the following PCR program:

94°C - 3 minutes

40 loops:

94°C-30 seconds

50℃-30 seconds

72°C-40 seconds

72°C - 5 minutes

4℃-permanent

Purified PCR product- PCR purification kit (Qiagen, cat. no. 28106)

a) Add 250 μl buffer PB to 50 μl PCR sample.

b) Place the QIA spin column in the provided 2ml collection tube.

c) To bind DNA, apply sample to QIA flash column and centrifuge for 30-60 seconds.

d) Discard the flow-through liquid. Put the QIA Flash Column back into the same tube.

e) For washing, add 0.75ml buffer PE to the QIA flash column and centrifuge for 30-60 seconds.

f) Discard the flow-through and place the QIA Flash Column back in the same tube.

g) Centrifuge the column for an additional 1 minute.

h) Place the QIA Flash Column in a clean 1.5ml microcentrifuge tube.

i) To elute the DNA, add 50 μl buffer EB (10 mM Tris·Cl, pH 8.5) or H ₂ O to the center of the QIA fast membrane, let the column rest for 1 minute and then centrifuge the column for 1 minute.

j) Measure the O.D. of the PCR product and dilute to create a 60 fg/μl stock solution.

3. Generate 1 ml of 1 μM Primer Mix A stock solution 1 by mixing 10 μl of 100 μM oligonucleotide stock solution (Table 2(a)) and adding water to 1 m. Mix well and then divide into 100 μl aliquots.

4. Create 1 ml of 1 μM primer mix B stock solution by mixing 10 μl of 100 μM oligonucleotide stock solution (Table 2(b)) and adding water to 1 ml. Mix well and then divide into 100 μl aliquots.

Multiplex PCR

1. Nucleic acid extraction - MasterPure ^™ DNA Purification Kit (Epicentre, cat. no. MC89010).

a) Add 100 μl of 1×PBS to 50 μl nasal rinse.

b) Add 150 μl 2×T&C lysis solution containing 1 μl proteinase K.

c) Incubate at 65°C for 15 minutes, vortexing every 5 minutes.

d) Incubate on ice or at 4°C for 3-5 minutes.

e) Add 150 μl MPC solution to the sample, and vortex to mix for 10 seconds.

f) Spin at maximum speed for 10 minutes.

g) Transfer the supernatant to a new 1.5 ml tube, then add 500 μl of isopropanol and mix well.

h) Centrifuge at maximum speed for 10 minutes at a4°C. The supernatant was discarded, followed by two washes with 80% ethanol.

i) The pellet was dried and resuspended in 8 μl of nuclease-free water.

2. Reverse transcription with primer LN-Invitrogen Superscript III (Invitrogen, catalog number 18080-093)

8 μl of NA from step 1

Primer LN (40μM) 1μl

5’-CGA TAC GAC GGG CGT ACT AGC GNN NNN NNN N-3’

10mM dNTP 1μl

TIM (60fg/μl) 1μl

NAC1 (60fg/μl) 1μl

           

Total volume 12μl

Incubate at 65°C for 5 minutes, then place on ice for over 1 minute Add the following reaction mixture to the tube and mix gently by aspiration:

5×First strand buffer 4μl

0.1M DTT 2μl

RNase OUT 1 μl

SuperScript III 1μl

           

Total volume 8μl

Run the following program on the PCR instrument:

25℃-10 minutes

50℃-50 minutes

85℃-5 minutes

3. Split multiplex PCR with primer L

a. Reaction A:

10×PCR buffer 5μl

50mM _MgCl2 4μl

50×dNTP 2μl

Primer L (100μM) 1μl

5’-CGA TAC GAC GGG CGT ACT AGC G-3’

Primer A mix (1μM) 2μl

5×Q-solution 5μl

RT template (from step 2) 10 μl

Platinum taq 2μl

Nuclease-free water 18 μl

UDG 1 μl

           

Total volume 50μl

b. Reaction B:

10×PCR buffer 5μl

50mM _MgCl2 4μl

50×dNTP 2μl

Primer L (100μM) 1μl

5’-CGA TAC GAC GGG CGT ACT AGC G-3’

Primer B mix (1μM) 2.5μl

5×Q-solution 5μl

RT template 10 μl

Platinum taq 2μl

RNase-free water 17.5 μl

UDG 1 μl

           

Total volume 50μl

Run the following PCR program:

94°C - 3 minutes

5 loops:

94°C-30 seconds

50℃-90 seconds

72°C - 2 minutes

35 loops:

94°C-30 seconds

64°C - 2 minutes

72°C - 5 minutes

4℃-permanent

Array preparation

Tag IQ-EX PCR-1.0kb Tag IQ-EX or 7.5kb Tag IQ-EX

1.0kb Tag IQ-EX PCR

Forward primer (1kb) 3μl

Reverse primer 3μl

Tag IQ-EX 5μl

_MgCl2 (50mM) 5μl

dNTP(10mM) 2μl

10×PCR buffer 10μl

Platinum Taq DNA polymerase 1μl

Water 71μl

             

Total volume 100μl

94°C, 3 minutes

30 cycles: 94°C, 30 seconds; 68°C, 30 seconds; 72°C, 40 seconds

·72°C, 10 minutes

7.5kb Tag IQ-EX PCR

Forward primer (7.5kb) 3μl

Reverse primer 3μl

Tag IQ-EX 5μl

dNTP(LA PCR Kit) 16μl

10×PCR buffer (LA PCR kit) 10μl

TaKaRa Taq 1 μl

Water 62μl

             

Total volume 100μl

94°C, 3 minutes

30 cycles: 94°C, 30 seconds; 68°C, 7 minutes 30 seconds

·68°C, 10 minutes

PCR纯化试剂盒(Qiagen，目录号28106)Purified PCR products (Tag IQ-EX and multiplex PCR)-

PCR purification kit (Qiagen, cat. no. 28106)

a) Add 500 μl buffer PB to 100 μl PCR sample (combine reactions A and B).

b) Place the QIA Fast Spin Column in the 2ml collection tube provided.

c) To bind DNA, apply sample to QIA flash column and centrifuge for 30-60 seconds.

d) Discard the flow-through liquid. Put the QIA Flash Column back into the same tube.

e) For washing, add 0.75ml buffer PE to the QIA flash column and centrifuge for 30-60 seconds.

f) Discard the flow-through and place the QIA Flash Column back in the same tube.

g) Centrifuge the column for an additional 1 minute.

h) Place the QIA Flash Column in a clean 1.5ml microcentrifuge tube.

i) To elute the DNA, add 40 μl buffer EB (10 mM Tris·Cl, pH 8.5) or H ₂ O to the center of the QIA fast membrane, let the column stand for 1 minute and then centrifuge the column for 1 minute minute.

j) Measure the O.D. of the PCR product.

Fragmentation and Tagging

1. Set up one tube per sample for fragmentation, and for each reaction, add EB buffer to a final volume of 35 μl. Handle Tag IQ-EX as a sample

line 1 Total μg of product added to array 1.4 μg line 2 The amount of fragmentation reagent U required for fragmented DNA Row 1 × 0.15 = 0.21 U line 3 Activity of Fragmentation Reagent (U/μl) 3 U/μl line 4 Volume of fragmentation reagent required for each reaction (μl) row 2 ÷ row 3 = 0.07 μl line 5 Volume of water required for each reaction (μl) 3.3 - row 4 = 3.23 μl

2. Preparation of Fragmentation Mixture on Ice

10× fragmentation buffer 4.3μl

Water 3.23 μl

Fragmentation Reagent 0.07μl

             

Total volume 7.6μl

3. Freeze the fragmentation mixture on ice, then add 7.6 μl to each DNA prepared from steps 1 and 2.

4. Run the following program:

37°C, 5 minutes

95°C, 10 minutes

4°C, keep

5. Preparation of Labeling Mixture (per reaction)

Tdt buffer (5×) 12μl

Gene Chip DNA Labeling Reagent (5mM) 2μl

TdT(30U/μl) 3.4μl

             

Total 17.4μl

6. Add 17.4 [mu]l labeling mix to each reaction and to the control fragmented PCR product.

7. Run the following program.

·37°C, 30 minutes

95°C, 5 minutes

4°C, keep

hybridize

1. Turn on the hybridization oven set at 45°C and warm the chip to room temperature.

2. Prepare the prehybridization solution.

1% Tween-20 2μl

1M Tris, pH7.8 2μl

Water 196μl

             

Total volume (per chip) 200μl

3. The chip is pre-hybridized with the pre-hybridization buffer at 45°C.

4. Form the hybridization master mix.

Tag IQ-EX ^* (fragmented 0.26μg) 1.9 μl

5M TMAC 132μl

1M Tris, pH7.8 2.2μl

1% Tween-20 2.2μl

Herring sperm DNA (10mg/ml) 2.2μl

Acetylated BSA 2.2μl

Control oligonucleotide B2 3.4μl

Water 13.9μl

             

Final volume (per chip) 160μl

^* Use the calculation below to determine how much fragmented Tag IQ-EX to use in the hybridization master mix.

Concentration of purified PCR product (eg 100ng/μl)×35μl=3500ng

The final volume after fragmentation and labeling is 60 μl

The final concentration of fragmented Tag IQ-EX = 58.3ng/μl

· 260/58.3 = 4.4658.3 μl will be required

5. Add 160 μl to 60 μl of labeled sample. Run the following program.

95°C, 5 minutes

45°C, 5 minutes

6. Remove prehybridization buffer from chip and fill with hybridization mixture.

7. Hybridize overnight at 45°C, 60 rpm.

washing and dyeing

1. Preparation of Wash Buffers A and B

Lotion A

20×SSPE 300ml

10% Tween-20 1ml

Water 699ml

           

Filter through a 0.2 μm filter and store capped at room temperature.

Lotion B

20×SSPE 30ml

10% Tween-20 1ml

Water 969ml

           

Filter through a 0.2 μm filter and store capped at room temperature.

2. Preparation of SAPE staining solution (per chip)

20X SSPE 360μl

1% Tween-20 12μl

50mg/ml acetylated BSA 50μl

SAPE 12μl

DI water 766μl

             

Mix well and divide into 2 aliquots of 600 μl (staining solution 1 and staining solution 3)

3. Preparation of antibody solution (per chip)

20×SSPE 180μl

1% Tween-20 6μl

50mg/ml acetylated BSA 25μl

10mg/ml normal goat IgG 6μl

0.5mg/ml biotinylated antibody 3.6μl

DI water 379.4μl

             

Final volume 600μl

4. Run Wash Protocol - DNA Array WS4.

Unlike conventional methods, the optimized RPM v.1 assay not only identifies the pathogen, but also provides sequence information, allowing detection and phylogenetic classification of a large number of pathogens within the same assay. Sequence information confirmed the ability of RPM v.1, a powerful tool for genetic variation analysis of circulating and emerging viruses (ie, influenza viruses), to identify type-wide variants (eg, HAdV). This capability is also used to track changes in known variants. This use was clearly demonstrated in influenza virus A positive clinical samples, showing that from the A/A/Panama/2007/99 sample strain (before the 2003 flu season) to the A/Fujian flu season in 2003-2004 Lineage changes in the /411/2002 strains followed by the A/California/7/2004 strains during the 2004-2005 influenza season. Only one M gene (H1N1) sequence that is relatively conserved in influenza virus A is collaged on RPM v.1. However, M gene ProSeq was still able to detect homologous regions of completely different subtypes, allowing correct discrimination (Table 10). This M gene ProSeq would theoretically allow the detection of any other type of influenza virus that does not tile the antigenic HA and NA sequences on the array.

This study demonstrates that such a system can display excellent clinical sensitivity and specificity, the ability to resolve complex co-infections without loss of sensitivity, and that sensitivity is similar for all 26 targeted pathogens and potential biological warfare agents. In contrast to culture and PCR assays, this assay platform showed comparable detection sensitivity and specificity for HAdV and Influenza A (Table 7). This data supports the feasibility of using the RPM v.1 system as a diagnostic tool to correctly identify and type clinically relevant adenovirus and influenza A strains within direct clinical specimens in a manner that is very relevant to routine surveillance methods.

Obviously many modifications and variations of the present invention are possible in light of the above teaching. It is therefore to be understood that the invention as claimed may be practiced otherwise than as specifically described. Any reference to a part of authority in the singular, eg, using the articles "a", "an", "the" or "said" is not to be construed as limiting that part to the singular.

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<223>

<400>13

cgatacgacg ggcgtactag cgttctaacc gaggtcgaaa cg 42

<210>14

<211>42

<212> DNA

<213> Artificial

<220>

Primers

<223>

<400>14

cgatacgacg ggcgtactag cgctctggca ctccttccgt ag 42

<210>15

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>15

cgatacgacg ggcgtactag cggggaggtc aatgtgactg gt 42

<210>16

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>16

cgatacgacg ggcgtactag cggggcaatt tcctatggct tt 42

<210>17

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>17

cgatacgacg ggcgtactag cggtgaaccg ttctgcaaca aa 42

<210>18

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>18

cgatacgacg ggcgtactag cgccaatctt ggatgccatt ct 42

<210>19

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>19

cgatacgacg ggcgtactag cgcattgaca gaagatggag aagg 44

<210>20

<211>41

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>20

cgatacgacg ggcgtactag cgaagcacag agcgttccta g 41

<210>21

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>21

cgatacgacg ggcgtactag cgctgtggac cgtgaggata ct 42

<210>22

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>22

cgatacgacg ggcgtactag cgttggcggg tatagggtag agc 43

<210>23

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>23

cgatacgacg ggcgtactag cgttattcag cagcacctcc ttg 43

<210>24

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>24

cgatacgacg gg cgtactag cgggtggcag gttgaatact ag 42

<210>25

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>25

cgatacgacg ggcgtactag cgggctgata atcttccacc tcc 43

<210>26

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>26

cgatacgacg ggcgtactag cgctctcacg gcaactggtt taa 43

<210>27

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>27

cgatacgacg ggcgtactag cggacaggac gcttcggagt ac 42

<210>28

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>28

cgatacgacg ggcgtactag cgggcaacat tggcatagag gaag 44

<210>29

<211>40

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>29

cgatacgacg ggcgtactag cgggtggagt gatggcttcg 40

<210>30

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>30

cgatacgacg ggcgtactag cgagtgccat ctatgctatc tcc 43

<210>31

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>31

cgatacgacg ggcgtactag cggccgtgga gtaaatggct aa 42

<210>32

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>32

cgatacgacg ggcgtactag cgagtcttcc aagaccgtcc aa 42

<210>33

<211>41

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>33

cgatacgacg ggcgtactag cgatgtgacc accgaccgta g 41

<210>34

<211>41

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>34

cgatacgacg ggcgtactag cggttgctgg agaacggtat g 41

<210>35

<211>45

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>35

cgatacgacg ggcgtactag cgtctacccc tatgaagatg aaagc 45

<210>36

<211>45

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>36

cgatacgacg ggcgtactag cgggataggc agttgtgctg ggcat 45

<210>37

<211>41

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>37

cgatacgacg ggcgtactag cgtgagtgcc agcgagaaga g 41

<210>38

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>38

cgatacgacg ggcgtactag cgcaggaggt gaggtagttg aatc 44

<210>39

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>39

cgatacgacg ggcgtactag cgtcaaatcc tcgttgacag ac 42

<210>40

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>40

cgatacgacg ggcgtactag cgtgcactgt tgcctccatt ga 42

<210>41

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>41

cgatacgacg ggcgtactag cgacaggaat tggctcagat atg 43

<210>42

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>42

cgatacgacg ggcgtactag cgacatgatc tcctgttgtc gt 42

<210>43

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>43

cgatacgacg ggcgtactag cgtcgaggtt gccaggatat agg 43

<210>44

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>44

cgatacgacg ggcgtactag cgggactatg agatgcctga ttgc 44

<210>45

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>45

cgatacgacg ggcgtactag cgcaactatt agcagtcaca ctcg 44

<210>46

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>46

cgatacgacg ggcgtactag cgaagttggc attgtgttca gtg 43

<210>47

<211>41

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>47

cgatacgacg ggcgtactag cgtcatccag actgtcaaag g 41

<210>48

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>48

cgatacgacg ggcgtactag cgaaacagga aacacggaca cc 42

<210>49

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>49

cgatacgacg ggcgtactag cgctctatca tcacagatct cagc 44

<210>50

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>50

cgatacgacg ggcgtactag cgcatgagtc tgactggttt gc 42

<210>51

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>51

cgatacgacg ggcgtactag cgacaaagat ggctcttagc aaag 44

<210>52

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>52

cgatacgacg ggcgtactag cgacccagtg aatttatgat tagc 44

<210>53

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>53

cgatacgacg ggcgtactag cgaaaaccaa cccaaccaaa cc 42

<210>54

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>54

cgatacgacg ggcgtactag cggcacatca taattggggag tgtc 44

<210>55

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>55

cgatacgacg ggcgtactag cggctctctt ggcgttcttc ag 42

<210>56

<211>42

<212> DNA

<213> Artificial

<220>

Primers

<223>

<400>56

cgatacgacg ggcgtactag cgtcattacc agccgacagc ac 42

<210>57

<211>41

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>57

cgatacgacg ggcgtactag cgccgtcagc gatctctcca c 41

<210>58

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>58

cgatacgacg ggcgtactag cgcctgtcca ccactccttg tc 42

<210>59

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>59

cgatacgacg ggcgtactag cggcttgaaa gggcagttct gg 42

<210>60

<211>43

<212> DNA

<213> Artificial

<220>

Primers

<223>

<400>60

cgatacgacg ggcgtactag cgcaggtctc cgattgtgat tgc 43

<210>61

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>61

cgatacgacg ggcgtactag cgctctggtg tgtggtgctt ata 43

<210>62

<211>40

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>62

cgatacgacg ggcgtactag cgctcggcac ggcaactgtc 40

<210>63

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>63

cgatacgacg ggcgtactag cgatgtggat gacgtttagg ta 42

<210>64

<211>41

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>64

cgatacgacg ggcgtactag cgggttgatg gcagtcggta a 41

<210>65

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>65

cgatacgacg ggcgtactag cgaagaagag ttcatgacgg ac 42

<210>66

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>66

cgatacgacg ggcgtactag cgtggttgtt tggttggtta ttcg 44

<210>67

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>67

cgatacgacg ggcgtactag cgccgatgac ttatagtatt ga 42

<210>68

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>68

cgatacgacg ggcgtactag cgataatctt gatgccactt agc 43

<210>69

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>69

cgatacgacg ggcgtactag cggttcttca ggctcaggtc aatc 44

<210>70

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>70

cgatacgacg ggcgtactag cgacagcggt atgtactggt cata 44

<210>71

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>71

cgatacgacg ggcgtactag cgtgggaata gtgtgcgtat gc 42

<210>72

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>72

cgatacgacg ggcgtactag cgacatcacc gcgacgcagc aa 42

<210>73

<211>40

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>73

cgatacgacg ggcgtactag cggattccgc gatgccgatg 40

<210>74

<211>44

<212> DNA

<213> Artificial

<220>

Primers

<223>

<400>74

cgatacgacg ggcgtactag cgcgcccatg tatttagaga accg 44

<210>75

<211>40

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>75

cgatacgacg ggcgtactag cgccggcgtc gtgcgcgaaa 40

<210>76

<211>40

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>76

cgatacgacg ggcgtactag cgcagccacg tcagccagcc 40

<210>77

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>77

cgatacgacg ggcgtactag cggagcgaat atctggcaca cc 42

<210>78

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>78

cgatacgacg ggcgtactag cggggccagg tctagaacga at 42

<210>79

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>79

cgatacgacg ggcgtactag cgtggagtac aatggtctcg agc 43

<210>80

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>80

cgatacgacg ggcgtactag cgtttgcatg aagtctgaga acga 44

<210>81

<211>41

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>81

cgatacgacg ggcgtactag cgacggcatt acaacggcta g 41

<210>82

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>82

cgatacgacg ggcgtactag cgcatcttct ggtaatccct gttc 44

<210>83

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>83

cgatacgacg ggcgtactag cgacagcgtt caatctcgtt gg 42

<210>84

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>84

cgatacgacg ggcgtactag cgagagaatt gcgatacgtt acag 44

<210>85

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>85

cgatacgacg ggcgtactag cgccttacaa cctattgaca cctg 44

<210>86

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>86

cgatacgacg ggcgtactag cgacacgaga gctacctgca ga 42

<210>87

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>87

cgatacgacg ggcgtactag cgtttataca atatgggcag gg 42

<210>88

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>88

cgatacgacg ggcgtactag cgtcgtaagc tgttcttctg gtac 44

<210>89

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>89

cgatacgacg ggcgtactag cgtcattgct tgatgaaact gat 43

<210>90

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>90

cgatacgacg ggcgtactag cgttggatat tcaccgaaca ctag 44

<210>91

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>91

cgatacgacg ggcgtactag cgcttgtgga aatgagtcaa cgg 43

<210>92

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>92

cgatacgacg ggcgtactag cgaggtagct atatttcgct tgac 44

<210>93

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>93

cgatacgacg ggcgtactag cggagcgtct acgtcctggt ga 42

<210>94

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>94

cgatacgacg ggcgtactag cgcattggtt tcgctgtttt ga 42

<210>95

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>95

cgatacgacg ggcgtactag cgtggaagag tgagggtgga tac 43

<210>96

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>96

cgatacgacg ggcgtactag cgaataatcc ctctgttgac gaa 43

<210>97

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>97

cgatacgacg ggcgtactag cgaggagcaa tgagaattac acg 43

<210>98

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>98

cgatacgacg ggcgtactag cgctaagttc caatactctt gc 42

<210>99

<211>45

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>99

cgatacgacg ggcgtactag cggccggtac ttatgtatgt gcatt 45

<210>100

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>100

cgatacgacg ggcgtactag cgcatcattg gcggttgatt ta 42

<210>101

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>101

cgatacgacg ggcgtactag cggggaacat acgcttccag at 42

<210>102

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>102

cgatacgacg ggcgtactag cgttccacat tttgtttggg aaa 43

<210>103

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>103

cgatacgacg ggcgtactag cgccttatcc gactcgcaat gt 42

<210>104

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>104

cgatacgacg ggcgtactag cgcagtgtga ggttatgtgg tgga 44

<210>105

<211>41

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>105

cgatacgacg ggcgtactag cgttggttgc gcaattcaag t 41

<210>106

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>106

cgatacgacg ggcgtactag cgtgttgttc tttgtgcagg aga 43

<210>107

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>107

cgatacgacg ggcgtactag cgtcgtaatg ttagctgtat catc 44

<210>108

<211>44

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>108

cgatacgacg ggcgtactag cgtacattag ctgtccactt accg 44

<210>109

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>109

cgatacgacg ggcgtactag cggtgggtgg tggtcttaag ttt 43

<210>110

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>110

cgatacgacg ggcgtactag cgctggatat taccagtgtc att 43

<210>111

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>111

cgatacgacg ggcgtactag cgactgataa agggggagtgg ata 43

<210>112

<211>41

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>112

cgatacgacg ggcgtactag cgctcgcctt gctctttgag c 41

<210>113

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>113

cgatacgacg ggcgtactag cgggacacaa gccctctcta cg 42

<210>114

<211>43

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>114

cgatacgacg ggcgtactag cgtagatacg gttacggtta cag 43

<210>115

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>115

cgatacgacg ggcgtactag cgcatgggaa gctgttttga tg 42

<210>116

<211>42

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>116

cgatacgacg ggcgtactag cgcccgaaga attgttccaa tc 42

<210>117

<211>21

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>117

gaccaatcct gtcacctctg a 21

<210>118

<211>21

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>118

gtatatgagk cccatrcaact t 21

<210>119

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>119

tcggtgggaa agaatttgac 20

<210>120

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>120

ttcctgatag gggctctgtg 20

<210>121

<211>24

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>121

acaagcaagg agatagcata gatg 24

<210>122

<211>24

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>122

gtaggagaag gtgtatgagt tagc 24

<210>123

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>123

acctgggcta attgggattc 20

<210>124

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>124

aatgcctgtt ggagcttgtt 20

<210>125

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>125

ggcttatgtg gccccttact 20

<210>126

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>126

aagatggccg cgtattattg 20

<210>127

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>127

ggctcagata tgcgagaaca 20

<210>128

<211>21

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>128

ttggtccggg taataatgag a 21

<210>129

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>129

ccatatgcgg catttataccc 20

<210>130

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>130

gcagtctctc tgcgttttcc 20

<210>131

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>131

tgctttaccc aaggcaaaaa 20

<210>132

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>132

agcctcatct gccaggtcta 20

<210>133

<211>23

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>133

aagatggctc ttagcaaagt caa 23

<210>134

<211>22

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>134

tactatctcc tgtgctccgt tg 22

<210>135

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>135

tggggcaaat acaaagatgg 20

<210>136

<211>23

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>136

cacatcataa ttgggagtgt caa 23

<210>137

<211>21

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>

ccaaccaaac aacaacgttc a 21

<210>138

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>138

accttgactg gaggccgtta 20

<210>139

<211>32

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>139

tcaactcgaa taacggtgac ttcttaccac tg 32

artificial

<210>140

<211>22

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>140

tgcagagcgt cctttggtct at 22

<210>141

<211>25

<212>DNA

<213>

<220>

Primers

<223>

<400>141

ctcttactcg tggtttccaa cttga 25

<210>142

<211>24

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>142

tggcgcccat aagcaacact cgaa 24

<210>143

<211>25

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>143

aacagatgta agcagctccg ttatc 25

<210>144

<211>26

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>144

cgatttttat tggatgctgt aattt 26

<210>145

<211>26

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>145

tgccatagca tgacacaatg gctcct 26

<210>146

<211>21

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>146

cggcagcgtc tcaattcgtt c 21

<210>147

<211>20

<212>DNA

<213> Artificial

<220>

Primers

<223>

<400>147

caagccgcct tcctcatagc 20

Claims (22)

1. A method comprising: Provide a biological sample suspected of containing nucleic acid from one or more pathogens within a predefined group of pathogens; Add a variety of PCR primers to the sample corresponding to the genes found in the predefined pathogen group; Wherein, the primers include at least one primer pair for each pathogen; wherein said primers comprise a trailer sequence that is not homologous to the DNA of any pathogen of a predefined group of pathogens or to any background DNA within the sample; and performing a polymerase chain reaction on the sample to amplify a subpopulation of nucleic acid corresponding to the gene; Wherein, the concentration of at least one primer in the polymerase chain reaction is not greater than about 100 nM. 2. The method of claim 1, wherein the concentration of each primer in the polymerase chain reaction is no greater than about 100 nM. 3. The method according to claim 1, wherein the tail sequence is CGATACGACGGGCGTACTAGCG (SEQ ID NO.1). 4. The method according to claim 1, wherein the trailing sequence is CGATACGACGGGCGTACTACTAGCGNNNNNNNNN (SEQ ID NO.2). 5. The method of claim 1, further comprising: extracting nucleic acid from a clinical sample to produce a biological sample. 6. The method of claim 5, wherein the clinical sample comprises a nasal rinse, a throat swab, sputum, blood or an environmental sample. 7. The method of claim 5, wherein the clinical sample is from an organism; and the trailing sequence is not of the same origin as the DNA of the organism species. 8. The method of claim 7, wherein the organism is a human. 9. The method of claim 1, wherein the pathogen is a respiratory pathogen. 10. The method of claim 1, wherein the pathogen is an enteric pathogen or a biological threat. 11. The method of claim 1, wherein the amplified nucleic acid comprises sequences of less than 200 nucleotides and sequences of greater than 2000 nucleotides. 12. The method of claim 1, wherein the trailing sequence reduces the formation of primer dimers. 13. The method of claim 1, wherein the PCR primers comprise at least 50 different primers. 14. The method according to claim 1, wherein, adding multiple PCR primers and carrying out polymerase chain reaction is carried out on multiple aliquots of biological samples respectively; wherein a different plurality of PCR primers are used for each aliquot; and Here, aliquots were pooled after the PCR reaction. 15. The method of claim 1, further comprising: contacting the sample with a microarray comprising a plurality of nucleic acid sequences that are at least partially complementary to the amplified nucleic acid; and The amplified nucleic acid is allowed to hybridize to a complementary nucleic acid. 16. The method of claim 15, wherein the complementary nucleic acid is a 25-mer to a 29-mer. 17. The method of claim 15, wherein the complementary nucleic acids comprise perfect match probes to at least one amplified nucleic acid and three different SNPs at the central position of each perfect match probe attitude. 18. The method according to claim 0, the method further comprising: It is detected which of the amplified nucleic acids hybridizes to the complementary nucleic acid. 19. The method of claim 0, further comprising: Pathogens are identified based on which amplified nucleic acid is detected. 20. The method of claim 19, wherein the identification is based on pattern recognition. 21. The method of claim 19, wherein the identification is based on sequencing of the amplified nucleic acid. 22. The method of claim 18, wherein said identifying comprises the strain of the pathogen.