AU650628B2 - A hybridization assay for campylobacter rRNA - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

650 Class Int. Class ApplicatiorrNumber: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Applicant(s): Ciba Corning Diagnostics Corp.

63 North Street, Medfield, Massachusetts, 02052, UNITED STATES OF

AMERICA

Address for Service is: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Complete Specification for the invention entitled: A HYBRIDIZATION ASSAY FOR CAMPYLOBACTER rRNA Our Ref 191129 POF Code: 1602/17948 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 600- 1 6006 A HYBRIDIZATION ASSAY FOR CAMPYLOBACTER rRNA BACKGROUND OF INVENTION 1. Field of Invention The present invention generally relates to oligonucleotide probes and to immunochemical techniques, and of methods of using such probes in combination with immunochemical techniques for diagnostic and other applicable purposes. More specifically, the invention consists of a sandwich hybridization assay to detect the presence of one or more Campylobacter species; and in an expanded assay format, the detection of one or more target nucleic acid sequences in a test sample, 2. Setting for the Invention: The genus Campylobacter is now recognized as a major cause of acute bacterial enteritis. These spiral-shaped pathogens have been shown to colonize the surface of the intestinal track in humans; and although the disease is self-limiting, early antibiotic therapy reduces the duration of the disease and fecal excretion. Since the discovery that Campylobacter is a cause of enteritis, a large number of strains of this bacteria have been identified by biochemical methodologies; however, the mechanism by which these species cause disease is still unknown. Studies examining the determinants of virulence attributed to Campylobacter include colonization, adhesion, invasion, and cytotoxin and endotoxin production. Labigne-Roussel et al, 170 p. 1704-1708, (1988).

The major medically important enteric pathogens are: C.

jejuni, C. coli, and C. laridis.

solid supports for hybridization assays have included nitrocellulose and chemically treated paper. Southern, J. Mol.

Bio. 98 p. 503-517 (1975). Applications of such hybridization techniques involve the use of natural or synthesized labelled nucleic acid molecules (probes) which are specific to aberant nucleic acid molecules and to etiologic pathogens.

Techniques for synthesizing nucleic acid hybridization probes consisting of sequences of deoxyribonucleotides or ribonucleotides are well known in the art. Typically, to construct a probe, a target DNA is isolated from a cell and denatured to form a single strand and copies of a portion of the strand are isolated or synthesized in a laboratory and then labelled. When exposed to a complementary strand of target DNA or RNA in a test sample, the labelled probe hybridizes its complementary target DNA or RNA sequence. Probes may be labelled using radioactive isotopes, fluorescing molecules, luminescent molecules, enzymes or immunochemical molecules. The label on the probe is then detected and the presence of target DNA or RNA of interest is thus detected.

Rashtchian, European patent application 87300569.8, describes oligonucleotide sequences which hybridize Campylobacter 16S rRNA. A labelled (avidin) solid support (plastic tube) quantitation assay is disclosed; where a labelled (biotin) DNA probe hybridized to Campylobacter 16S rRNA is detected by measuring unbound label on the solid support (the amount of biotin bound to the tubes being estimated by absorbence after treatment of the the tubes with avidin).

-3- Goodson, European Patent Application 87302354.3, describes a colormetric liquid hybridization assay for detecting nucleic acid sequences using at least two labelled oligonucleotide probes; and then capture of the hybrid complex to a solid support (microtiter well) for separation. Methods are described for detecting a restriction site characteristic of sickle cell anemia and for detecting the pilus surface antigen for N.

gonorrheae.

Hogan et al, European patent application 87310363.4 describes a method for preparing oligonucleotide probes complementary to a variable -gion of rRNA, selected to be unique to non-viral organisms. Probes specific to Campylobacter 16S rRNA are disclosed.

Rashtchian et al, U.S. Patent 4,785,086, describes DNA probes (900-1500 nucleotides) that are capable of hybridizing DNA of at least 80% of bacteria of in the species Campylobacter jejuni. Denatured bacterial DNA is immobilized on a binding support, the bacterial DNA is then hybridized by a labelled S probe. Another method is cited which describes the use of unlabelled probes; where the contacting and detecting steps are performed by sandwich hybridization.

Law et al, U.S. 4,745,181 describes the use of chemiluminescent labels in specific binding assays such as immunoassays or nucleic acid hybridization assays. A polysubstituted aryl acridinium ester is disclosed.

-4- Josephson, U.S. Patent 4,672,040, describes the use of magnetically responsive particles in nucleic acid hybridization assays. Nucleic acid coupled magnetic particles are dispersed in a reaction mixture containing molecules to be isolated, allowing hybridization and then separating the particles with the bound molecules from the reaction mixture. The hybridized mol'cule can then be separated from the magnetic particle.

Hill et al, International Application PCT/GB86/00176 discloses the use of magnetic or magnetizable substance, coated with a material capable of attachment to single-stranded DNA or RNA for separation purposes. Fowlowing separation the material linked to the magnetic or magnetizable substance is contacted with a probe to detect the presence of the single-stranded material by hybridization.

Rashtchian et al, Clin. Chem. 33/9 p. 1526-1530 (1987), describes an immunological capture method for nucleic acid hybrids and its application to nonradioactive labelled DNA probe assays. Synthetic DNA probes complementary to Campylobacter 16S rRNA were labelled with biotin and then hybridized to ribosomal RNA from lysates of bacterial cells. After hybridization, the hybrids were captured with immobilized anti-DNA:RNA antibody and the biotinylated probe was detected with streptavidin-horseradish peroxidase conjugate. The assay was optimized to detect 70,000 Campylobacter cells from a pure culture sample.

Heller et al, European patent application 82303701.5, describes a light-emitting polynucleotide hybridization assay.

A solid support method is described which comprises the steps of immobilizing a target single-stranded polynucleotide on a suitable support; contacting the immobilized sample with a labelled (peroxidase or iron porphrin derivative) single-stranded polynucleotide segments which are complementary to the target single-stranded polynucleotide; separating unhybridized single-stranded polynucleotide segments; exposing the immobilized hybrid to means for exciting the light label; and detecting the light response.

Hansen, European Patent Application 84306513.1, describes a sandwich hybridization assay including the formation of a biotinylated nucleic acid probe bound to an avidin coated solid support and reacting the solid support bound probe with the hybridization product of an enzyme labelled nucleic acid and a target nucleic acid.

Ranki et al., U.S. Patent 4,563,419 describes a competitive S one-step sandwich hybridization assay for detection of target microbial nucleic acids. A first nucleic acid probe affixed to a solid support is hybridized with target nucleic acid sample i and a labelled second nucleic acid probe. Following hybridization the label associated with the hybrid complex bound to the solid support is detected.

Malcolm, International Application PCT/GB85/00591, describes a sandwich hybridization reaction (two overnight incubations) utilizing solid support (polymer beads) as a carrier for an immobilized nucleic acid fragment and a non-immobilized second labelled nucleic acid.

Soderlund, UK Patent 2,169,403, recites a solution hybridization method for identification of nucleic acide. A detection (radio-label) nucleic acid probe and a capture nucleic acid probe are hybridized with the target nucleic acid sequence prior to capture to the solid support (affinity chromatography column).

Snitman, International Application PCT/US86/01280, describes a solution hybridization assay including immobilizing the resultant hybrid complex on a solid support, and followed by a second hybridization of the target nucleic acid sequence. A modified method utilizes a distinct second probe during the first hybridization step in order to increase the capture to the solid support.

Kohne, U.S. Patent 4,851,330, describes a method of detecting, identifying, and quantitating a group of non-viral organisms by hybridization assay.

Other patents which may be considered to be of interest include (cited in alphabetic order): Chiswell, U.S. Patent 4,716,106 entitled: "Detecting Polynucleotide Sequences"; -7- Daltagupta, U.S. Patent 4,670,380 entitled: "Assays Utilizing Labelled Nucleic Acid Probes"; Gingeras et al, International Application PCT/US87/01966 entitled: "Nucleic Acid Probe Assay Methods and Compositions"; Heller, European patent application 86118191.5, entitled: "Method Ior Increasing the Sensitivity of Nucleic Acid Hybridization Assays"; Kourilsky et al, U.S. Patent 4,581,333 entitled: "Method of Detecting a Nucleic Acid or Reactant for the Application of this Method"; Miller, European patent application 85309224.5, entitled: "Polynucleotide Hybridization Assays Employing Catalyzed Luminescence"; Nogueira et al, U.S. Patent 4,801,530 entitled: "Nucleotide Hybridization Assay for Protozoan Parasites"; Rabboni et al, European patent application 85105130.0 entitled: "Hybridization Method for Detection of Genetic Materials"; Stabinsky, U.S. patent 4,797,355 entitled: "Methods for Attaching Polynucleotides to Supports"; Taber et al, U.S. Patent 4,689,295 entitled: "Test for Salmonella".

-8- There remains still a need in the art for a timely simple and sensitive method for detecting one or more bacteria genera or species, which utilizes the rapid kinetics of liquid hybridization and also allows the hybridized products to be separated from unhybridized probes and debris of the test sample.

DEFINITIONS

The following terms, as used in this disclosure and claims, are defined as: 1. Bacteria: members of the phylogenetic group eubacteria, which is considered to be one of the three primary kingdoms.

2. Complementarity: a property conferred by the base sequence of single strand of DNA or RNA which may form a hybrid or a double-stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick base pairs on the respective strands. Adenine usually complements Thymine (T) or Uracil while Guanine usually complements Cytosine 3. Hybrid: the complex formed between two single stranded nucleic acid sequences by Watson-Crick base pairing or non-canonical base pairings between the complementary bases.

4. Hybridization: the process, environment and conditionsunder which at least two complementary strands of nucleic acids combine (anneal) to form a double-stranded molecule (hybrid).

Kit: a packaged combination of containers holding the necessary assay components for performing the sandwich hybridization method to detect the presence of one or more -9target nucleic acid sequences in a test sample. Apparatus, instrumentation devices and standard reagants necessary for performing the assay may or may not be a component(s) of the kit.

6. Liquid hybridization: refers to a hybridization of one or more nucleic acid probes and a target nucleic acid sequence in a liquid medium, without the presence of any solid support.

7. Mutually exclusive region: means that under hybridization conditions of the preferred embodiment the two probes should not compete for the same nucleotide base sequence on the target to the extent that hybridization of one probe prevents the hybridization of other probe(s).

8. Nucleic acid probe: a single-stranded nucleic acid seqaence that will combine (anneal) with a complementary single-stranded target nucleic acid sequence to form a double-stranded molecule (hybrid). A nucleic acid probe may be an oligonucleotide.

9. Nucleotide: a subunit of nucleic acid consisting of a phosphate group, a 5 carbon sugar and a nitrogen-containing base. In RNA the 5 carbon sugar is bose. In DNA the 5 carbon sugar is a 2-deoxyribose.

Oligonucleotide: a nucleotide polymer generally about.

ten to fifty nucleotides in length.

11. Probe specificity: characteristic of a probe which describes its ability to distinguish between target and non-target nucleic acid sequences. Probe specificity may be absolute probe able to distinguish between target organisms and non-target organisms), or it may be functional probe able to distinguish between the target organism and any other organism normally present in a test sample). Many probe sequences may be adapted for use either broadly or narrowly depending upon the assay conditions of such use.

12. Sandwich immunoassay: involves coupling an antibody (monoclonal or polyclonal) directed to a first antigenic determinant to a solid support and exposing the solid support-coupled antibody to a test sample containing a substance bearing the first and a second antigenic determinant. This results in the removal of the antigenic substance from the sample by the formation of a primary antibody--antigen complex which is bound to the support. The complex is then exposed to a second labelled antibody directed toward a second antigenic determinant on the antigenic substance to create an antigen antibody sandwich which can be separated and detected. The sandwich assay may be modified to incorporate the use of other pairs of complementary molecules.

13. Target nucleic acid: refers to a segment of single-stranded polynucleotides having a nucleotide base sequence corresponding to a genetic element whose presence in a.

test sample is to be detected.

14. Test sample: refers to sample containing one or more target nucleic acids and which may be in purified or nonpurified form. Test samples may be obtained from any physiological or -11laboratory source, for example from cells, biological tissue extract, DNA or RNA (synthesized or natural) from any source including viruses, and the like.

It would be desirable to provide an assay for detecting and quantitating the presence of one or more Campylobacter species in a test sample, and one which affords a more timely, specific, and sensitive methodology over culture techniques.

It would be desirable to provide a test kit for detecting and quantitating the presence of one or more genus or species of bacteria in a test sample.

It would be desirable to provide a plurality of oligonucleotide probes which are complementary and specific to Campylobacter 16S rRNA.

It would be desirable to provide an oligonucleotide probe assay which does not require the use of-radioactive materials.

It would be desirable to provide an assay which wii allow quantitation of Campylobacter in a test sample having about 10,000 cells/ml of the bacterium.

It would be desirable to improve the detection of chemiluminescent labelled entities, and particular chemiluminescent labelled oligonucleotide probes.

It would be desirable to provide an assay for detecting the presence of one or more target nucleic acid sequences in a test sample, and one which affords a more timely, specific and sensitive methodology.

SUMMARY OF THE INVENTION The present invention provides a method for assayir 9 Campylobacter rRNA including: a. providing a test sample including cells of one or more cell types, said test sample including cells of one or more cell types, said test sample suspected of containing Campylobacter cells or Campylobacter rRNA, and wherein said Campylobacter cells or Campylobacter rRNA may include one or more species of Campylobacter; b. releasing rRNA from the cells of said test sample; Sc. hybridizing rRNA of Campylobacter, if present, in said test sample, with at least two labelled oligonucleotide probes to form a hybrid complex, each of said probes having a nucleotide sequence that is complementary and at least one of which is specific to a region of Campylobacter 16S rRNA, and wherein at least one of said probes is labelled with one or more first support binding partners, and wherein at least one of said probes is labelled with one or more detector 12 molecules; d. capturing said hybrid complex on a solid support ,to form a sandwich complex, said solid support having one or more second support binding particles immobilized thereon which are. complementary to said first support binding partners, and wherein said first support binding partners bind to said second support binding partners; e. isolating said sandwich complex from said test sample and excess nonhybridized probes, f. detecting the presence-of Campylobacter by the activation of detector molecules associated with said sandwich complex; and g. quantitating the number of Campylobacter cells in said test sample an wherein the sensitivity of said test assay permits quantitation of approximately 1 x 104 bacteria cells per milliliter of test sample.

The present invention further provides a test kit suitable for detecting and quantitating Campylobacter in a test sample including: a. a solution for releasing rRNA from Campylobacter cells; b. a first labelled oligonucleotide probe, said probe being complementary to Campylobacter rRNA, and wherein said label is a first support binding partner; c. a second labelled oligonucleotide probe, said probe being complementary to Campylobacter rRNA, wherein said label is a detection molecule; and wherein first probe or said second probe is specific to a region of Campylobacter 16S rRNA; d. a solid support, said solid support having a second support binding partner bound thereto which is complementary to sa:d first support binding partner.

t56 The present invention further provides a method for assaying the presence of one or more target nucleic acid sequences in a test sample including: a. providing a test sample including cells of one or more cell types, said test sample suspected of containing one or more target nucleic acid sequences; S b. releasing target nucleic acid sequences from the cells of said test sample; c. hybridizing the target nucleic acid sequence, if present in said test sample, with a plurality of distinct oligonucleotide probe units to form a plurality of hybrid complexes, each of said probe units including at least two labelled oligonucleotide probes, each being complementary to and at least one OT which 13 is specific to a region of the target nucleic acid and wherein at least one of the probes of said unit is labelled with one or more first support binding partners, t~re...

and wherein at least one of the probes of said unit is labelled with one orLcowe distinct detector molecules; and wherein said distinct detector molecules are activated sequentially or simultaneously; d. capturing said hybrid complexes on a solid support to form sandwich complexes, said solid support having one or more second support bindingt1afies immobilized thereon which are complementary to said first support binding partners, and wherein said first support binding partners bind to said second support binding partners; e. isolating said sandwich complexes from said test sample and nonhybridized probes of said units; f. detecting the presence of one or more target nucleic acid sequences by the activation of the detector molecules associated with said sandwich complexes, and wherein each of said detector molecules provides a discernible activation reaction; and g. quantitating the number of bacterial cells of one or more genus or species in said test sample, and wherein the test assay permits quantitation of approximately 1 x 104 bacteria cells per milliliter of test sample.

The present invention further provides a test kit suitable for detecting and quantitating the presence of one or more target nucleic acid sequences in a test sample including: a. a solution for releasing target nucleic acid sequences from the cells; b. at least two distinct first labelled oligonucleotide probes, each of said probes being complementary to a target nucleic acid sequence and wherein said label is o. ~a first support binding partner; S c. at least two distinct second labelled oligonucleotide probes, each of said probes having a discernible label and being complementary to a target nucleic acid sequence and wherein each of said probes being complementary to a target nucleic acid sequence as is one of the first probes, and wherein said label is a detector molecule and wherein at least one of either said first probes or said second probes, complementary to the same target nucleic acid sequence is specific to a region- of the target nucleic acid sequence; and 0- 13a d. a labelled solid support, and wherein said label is a second support binding partner which is complementary to said first support binding partner.

In general, the invention consists of a sandwich hybridization assay for Campylobacter species. The assay is a two-step procedure, the first step involves a solution hybridization, which results in the formation of a hybrid complex comprising a target nucleic acid sequence and at least two labelled oligonucleotide probes, each probe being complementary and at least one probe being specific to a region of the target nucleic acid sequence. In the second step the hybrid complex is captured by a solid support. The label of at least one probe (ie. detector probe) being used for the detection of the target nucleic acid sequence; and the label of at least one probe (ie.

capture probe) being used to bind to the solid support. Only target nucleic acid sequences hybridized with both the detector and capture probes, and bound to the solid support are detectable in the described assay format.

o 0, t 13b The format of the assay may be expanded to incorporate a plurality of units of oligonucleotide probes, where each unit includes at least one capture probe and at least one detector probe. The capture and detector probes of each unit being complementary and at least one being specific to a region of the target nucleic acid sequence from a test sample. The detection and quantitation of one or more target nucleic acid sequences, for example one or more genus or species of bacteria, in a test sample is performed similarly to the described Campylobacter assay.

The preferred assay format incorporates a chemiluminescent molecule as the label of the detector probe; the chemiluminescent molecule being an acridinium ester. The chemiluminescent molecule reacts with appropriate reaction reagents to produce a light signal which enables the assay to provide a level of sensitivity to detect target nucleic acid sequences from a test sample which contains approximately 1 x 4 bacteria cells per ml of test sample.

The preferred assay of this invention further incorporates the use of solid support, labels, and devices of the Magic

R

Lite assay system (Ciba Corning Diagnostics Corp.); however, other solid supports, including Sepbarose 6B-CL, capture or detector labels, and devices can be employed. With such modifications, improvements in detection sensitivity can also be obtained in accordance 7ith the principles of this invention.

-14- BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the results of the comparative label assay for Campylobacter as described in Example 1.

Figure 2 illustrates the results of a MagicR Lite sandwich hybridization assay for Campylobacter rRNA as described in Example 2.

Figure 3 illustrates the results of a MagicR Lite sandwich hybridization assays varying the format of chemiluminescent labelling as described in Example 3.

DESCRIPTION OF PREFERRED EMBODIMENTS Oligonucleotide probes may be prepared synthetically, semisynthetically, by recombinant-DNA techniques, or from nucleic acids isolated from purified target nucleic acid sequence samples. Probes are also available from several sources, including Promega Corp., Madison, WI, U.S.A.

The present invention incorporates methods for the synthetic preparation of DNA oligonucleotide probes for use in hybridization assays of Campylobacter rRNA. See Applied Biosystems Model 380B DNA Synthesizer Users Manual, Version July, 1983. Applied Biosystems Model 381A DNA Synthesizer Users manual, Version 1.11, November, 1985. Beaucage et al, Tetrahedron letts. 22: 1859-1862 (1981). Matteucci and Caruthers, J. Am. Chem. Soc. 103: 3185-3191 (1981). Sinka et al., Tetrahedron Letts. 24: 5843-5846 (1983). These procedures may be utilized to prepare oligonucleotide probes for other target nucleic acid sequences including various genera or species of bacteria to be tested for in the expanded format of the assay.

Table 1 lists thirteen oligonucleotide probes specific and complementary for Campylobacter 16S rRNA. The first column provides an assigned alphanumeric designation for each probe (assigned by Promega Corp.), the second column shows the location of the probe corresponding to E. coli 16S rRNA which they hybridize, the third column shows the base length of the probe, and the fourth column shows the sequence of the probe.

It is noted that probes PM 78, PM 122 and PM 138 correspond to the same region of the E. coli 16S rRNA.

The probes of Table 1 were tested for species specificity by a hybridization procedure. A slot blot hybridization procedure was used to assay the specificity of the probes on a variety of purified RNA samples isolated from Campylobacter (n=133) and non-Campylobacter organisms See Kafatos et al, Nucl.

Acid Res. 7(6) p.1541-1552 (1970) and Tolsty et al, Cold Spring Harbor Laboratory Schimke, ed.) p. 231-238 (1982). The RNA samples were isolated according to the method listed in Table 2. Table 3 provides detailed hybridization results for each probe and each strain of micro-organism analyzed. A zero indicates lack of hybridization, a two indicates strong hybridization, a one indicates weak hybridization, and blank space indicates that no hybridization data was interpreted for.

that particular strain. Reference is made to the legend in Table 3. An abbreviated summary of the data contained in Table 3 is provided in Table 4. On the basis of analyzing the results of Table 3, at least two of said probes may be selected and incorporated for use in the Campylobacter assay of the -16preferred embodiment. For example, probe PM78 may be selected in one assay format based on its ability to detect C. fetus. In the same assay format probes PM 122 and PM 138 should not be selected as the second probe, since each may compete for the same 16S rRNA region as PM 78.

It is understood that many of the same strains of micro-organisms analyzed for probe specificity in Table 3 will comprise targets for oligonucleotide probe units to be incorporated in the expanded assay format as described below.

Similar species specific testing would be required before incorporating such oligonucleotide probe units into the expanded format.

After the oligonucleotide probes are tested for species specificity, the ends of individual probes are modified by chemical groups capable of forming a stable complex with an analog of the said chemical group. See Smith et al, Nucl. Acid Res. 13(7) p. 2399-2411 (1986) and Haralambidls et al, Nucl.

Acid Res. 15(12) p. 4857-4864 (1987). The chemical groups are chosen so as not to interfere with the hybridization step of the assay. In the described Examples and in the preferred embodiment at least one probe is labelled with one or more first support binding partner(s) and at least one probe is labelled with one or more detector molecule(s) by techniques, known in the art, including covalent bonding.

-17- The function of the support binding partners is to facilitate the capture (separation) of the hybrid complex to the solid support. The purpose and function of the solid support is described below. The hybrid complex comprises a target nucleic acid sequence annealed with at least two distinct oligonucleotide probes, with at least one of said probes having one or more first support binding partners bound thereto. The solid support in turn, includes one or more second support binding partners immobilized thereoa-, which have specific affinity to said first support binding partner.

The high affinity of biotin for avidin or strepavidin is well suited for their use as support binding partners in the present invention. U.S. Patent 4,582,810, describes the formation of avidin-biotin bridges in a diagnostic composition with binding to solid support particles. Murasugi et al, DNA 3(3) p. 269-277 (1984) describes the use of biotin labelled oligonucleotides as hybridization probes.

An immunoassay generally refers to a method of determining the presence or quantity of a substance in a test sample which method is based on the use of antibodies specific to that substance. The assay reaction requires the formation of an immunochemical complex between the antigenic substance (hapten)-.

and its respective antibody, this occurs by simply incubating the antibody with a test sample containing the antigen.

-18amita or the reaotants ot the iumunochemzical comple~x may be imobilized on a so lid support an provided iz ona of the embodiments WE this invention. if a hapteri is used as the first tiupport binding partner,, such as dinitrophenol (D2np) of ExamplQ 1, described below, then ar. antibody (preferable monioclonal) to DNP, and specifically anti-DNP (5H1) in that preferrad emfbodmenft, will serve as the complementary se .oiad support bindingj partner.

Niumerous non-radioactive and radioactive labelling techn-tques,, and detection protocols are known for detecting a hybridized complex. X detector 'molecule is typizcally chosen so that there is iminimal interference with the base pai.,ring (Watson-Crick) between the ol.igonuleotide probes and the target nucleic acid sequence. Examples of such molecules include radioactive, luminescent or fluorescent materials, etzymes. which create luxinescent, fluo~rescent, or colorometric products, and others as known in ths art.

In the preferred emobodimenit the detector molecule is a ceilumineicent molecule and more specifically an acridinium estar or a polysubstitaited acridiniam e"ter. The preparation and chemistry of polysubstituted aryl acridinium esters is described in U.S. Patent 4,745,181, and which is incorporated by 99re 9 0=6rnc. Tuef light emission (signal) clenarated by the activation of 'the ceiunescarit molecule may be detected by a cozmercially available instrameut, for example. a ~I instrument. (ciba Corning Diagnostio Corp.) One or more detector molecules may be bound to an oligonucleotide probe in order to enhance detection sensitivity. It is a requirement of this information that no first binding partners be bound to the probe labelled with the detector molecules. At least one oligonucleotide probe labelled with one or more detector molecules comprise one component of the oligonucleotide probe unit in the expanded assay .ormat of the present invention. Where a plurality of oligonucleotide probe units are incorporated into a desired assay format, more than one type of detector molecule per probe unit may be required to differentiate the signals or reactions generated on the activation of the detector molecules. If more than one detector molecule is employed, they may be detected sequentially in a manner as shown below in Example 1, or by use of chemiluminescen .:~bels having expression signals at distinct wavelengths. Alternatively, multiple detector molecules may be detected simultaneously.

The labelled oligonucleotide probes may now be utilized in a hybridization assay for a target nucleic acid sequence.

The test sample preparation requirements includes the lysis "1 of the organisms of interest and the protection of the released RNA from the nucleases found in the sample. An optimal lysis procedure would lyse the desired enteric pathogens without requiring that every cell in the specimen lyse.

Campylobacter cells were found to lyse in a Tris, EDTA buffer (pH 8-9) which contains SDS in the range of 0.05% to at room temperature. Cells remain viable in the Tris, EDTA buffer in the absence of SDS. A lysis time of 1 to 10 minutes was acceptable. Shigella and Salmonella cells were more resistant to lysis than Campylobacter. Acceptable conditions for the quantitative lysis of these organisms required either a higher SDS concentration or a higher temperature.

Lysis in 0.25% SDS in a Tris, EDTA buffer (pH 8-9) for minutes at room temperature was the preferred procedure for cultured Shigella, Salmonella, and Campylobacter cells. This procedure, however, does not protect the released RNA in a stool specimen.

In order to recover released RNA from a stool specimen other steps are required. The preferred method is to combine SDS lysis with a heat inactivation step, 75°C for 10 minutes) followed with filtration (LID/x (Genex) 25 um polyethylene).

Other nuclease inhibitors were examined, but none other than the heat inactivation with SDS are required. For example, the S inclusion of 5 to 10 mM VRC (vandyl ribonucleoside complex) with lysis at 65 0 C stabilizes the RNA as well as a lysis at 75 0

C

S without VRC.

-21- In the present invention each of the oligonucleotides are specific and complementary and at least one of which is specific to a target nucleic acid seqence suspected of being present in the test sample. The synthetic oligonucleotide probes may be both complementary and specific, for example, to any one of the variable regions of the rRNA, comprising the 5S, 16S or 23S rRNA. In the described method for detecting and quantitating the presence of Campylobacter species in a test sample, the oligonucleotide probes, as listed in Table 1, are both specific and complemertary to mutually exclusive regions of Campylobacter 16S rRNA.

Hybridization conditions necessary to accomplish annealing of the target nucleic acid sequence, if present, and at least two distinct oligonuclectide probes are determined by a number of variables, many of which may be adjusted to enhance the efficiency of the assay. The variables include: the nature of the labels attached to the probe, the sequence of the probe, the size (base pairs) of the probe, method of release/preparation of S target nucleic acid sequence, and the duration and temperature Ioo S of hybridization. It is also acknowledged that technical experience influences the efficiency of test assay. It is one aspect of this invention that these variables be reduced or made more uniform by providing a test kit which utilizes packaged assay components and the use of such kit components in combination with automated instrumentation (MLA system, Ciba Corning Diagnostics Corp.).

-22- Immobilization and separation of the hybrid complex from test sample debris and excess unhybridized probe is accomplished by use of a solid support. It is one of the advantages of a solid support that a plurality of binding partners can be immobilized thereon. Solid supports which have been utilized in hybridization assays include plastic tubes, microtiter wells, cross-linked dextran, porous silicate glass, magnetic particles coated with cellulose derivatives, nitrocellulose filter and other known materials which are inert to the assay components.

In the preferred embodiment paramagnetic particles (PMP) are utilized as the solid support. The paramagnetic particles are of the type disclosed in U.S. Patent 4,554,088.

The immobilization of the second support binding partner on the PMP may be accomplished by a number of techniques depending on the characteristics of the second support binding partner.

In the case where the second support binding partner is an antibody, the antibody may be coupled to PMP by utilizing glutaraldehyde as a coupling agent. See Reichlin, Method of Enzy., 70 p. 159-165 (1980).

In the preferred embodiment the second support binding S partner functions to capture the hybrid complex to the solid support. The complex is then separated from test sample debris.

and excess unhybridized probe by use of a magnetic field. (MLA rack, Ciba Coming Diagnostics Corp.) Alternate methods of separating the sandwich hybrid complex are described in the art, however, the MLA rack provides the advantage of processing a plurality of test samples simultaneously.

-23- EXAMPLE 1 Varying amounts of Campylobacter jejuni cells (ix10 4 to 4x10 6 of a known source, ATCC #29428, were added to 100 ul of a 1:50 dilution of feces containing 50 lM Tris (Sigma Chemical pH 9.0, 0.6 M NaCi (Sigma Chemical 60 mM sodium citrate (Mallinckrodt), pH 7.5 and 0.05% SDS (Sigma Chemical The cells of the sample preparation were lysed by heating to 950 C for 5 minutes. The released rRNA was hybridized for hrs. at 56 0 C with 0.9 picomoles (pmol) labelled oligonucleotide probe (DNP-PM78, DNP labelled at its 5' end)) and 0.2 pmol labelled oligonucleotide "probe (PM238), labelled with a chemiluminescent material (acridinium ester at its terminus and 32 P at its 3' terminus.

The nucleotide sequence of PM78 comprises: 5'-TCT GCC TCT CCC TCA CTC TAG ACT ATG AGT T-3'.

The nucleotide sequence of PM238 comprises: 5'-GCC TTC GCA ATG GGT ATT CTT GGT GAT-3'.

Following hybridization, 10 ul aliquots were added to 50 ug of paramagnetic particles (PMP) to which anti-DNP antibodies (mouse monoclonal) had been covalently attached. After 0.50 p.

hrs. of incubation at room temperature (230 the bound sandwich hybrid was separated from excess unhybridized probes and test sample by separating the PMP in a MagicR Lite rack, S (Ciba Corning Diagnostics Corp.) then the supernatant was removed. The PMP were washed twice with 0.6 M NaCl, 60 mM sodium citrate, 10 mM Tris, pH 7.0, 50 mM EDTA, (Sigma chemical -24- 0.1% bovine serum albumin (BSA) (Miles, fraction V) and 0.02% Tween-20 (Sigma Chemical Co.) and then resuspended in 100 ul of distilled water. It is noted that the solid support will also capture unhybridized probes labelled with the capture molecule but will not capture unhybridized probes labelled with the detector molecule. The bound sandwich hybrids were detected by a chemiluminescent reaction expressed in relative light units (RLU) in a MagicR Lite analyzer, (Ciba Corning Diagnostics Corp.) using modified reaction reagents (Reagent 1 comprising N HNO 3 in a 0.5% solution of H 2 0 2 Reagent 2 comprising 2.5N NaOH in a 0.5% solution of surfactant (Arquad)) (See also Example 6) (Ciba Corning Diagnostics Corp.) as well as liquid scintillation counting (atom light liquid scintillation cocktail, New England Nuclear) of the samples. Campylobacter rRNA was detected above background at cell counts in the order of 10,000 Campylobacter cells in a test sample. Figure 1 provides a comparison of the two labels relative to the concentration of Campylobacter cells in a test sample. As a control, purified E. coli rRNA (Pharmacia) were hybridized, captured and detected in the same manner (except in the abseice of feces) and did not give light signals above background signal. The background signal was the chemiluminescent reaction observed (RLU expressed) from samples treated in the same manner but whose binding to be labelled PMP was blocked by the addition of beta-analine DNP. The conversion of the signal to cell counts was calculated by the following formula: r i ~II pls~ ll-l, -~Pn The concentration of Campylobacter jejuni cells in suspension was determined by quantitative culture or fluorometric assay with a bibenzimidazole dye (Hoechst 33258).

Cell suspensions were diluted 106 and 108 fold in ice cold sterile water. Samples (10 and 100 ul) of dilutions were innoculated onto Brucella agar with 5% horse blood or Trypsoy agar with 10% sheep blood. Plates were incubated at 370 C in a microaerophilic environment 02, 10% CO 2 and

N

2 Colonies were counted after 48 hours of incubation.

Fluorometric determination of the DNA concentration of Campylobacter lejuni suspensions was made using Hoechst 33258 dye. Concentration of DNA in the suspension was determined by incubation of the cell suspension and dye for 0.50 hrs. at room temperature (23 0 C) in the dark. Fluorescent readings were compared to a lambda DNA standard curve. Calculation of the number of cells/ul was based upon the genome size of C. iejuni: 1) 2.2 x 109 g/mole 3.65 x 10 15 g DNA/cell 6.02 x 10 molecules/mole 2) measured g DNA/uL No. cells/uL 3.65 x 10-1 5 g DNA/cell EXAMPLE 2 Various amounts of Campylobacter rRNA (100 attomoles (amol)- 100 fentomoles (fmol) were suspended in 500 ul of 0.6 M Sodium Chloride, 60 mM sodium citrate, 10 mM Tris, pH 8.0, 50 mM EDTA and 0.05% SDS were hybridized at 56 0 C for 2.0 hrs. with 0.25 pmol labelled oligon-cleotide probe (AE-PM77), the probe being -26labelled with a chemiluminescent molecule (AE) at its terminus, and 0.5 pmol labelled oligonucleotide probe (AE-PM238), the probe being labelled with a chemiluminescent molecule (AE) at its 5' terminus and 1.0 pmol labelled oligonucleotide probe (biotin-PM78 labelled at its 5' terminus, the probe being labelled with a first support binding partner (biotin source Aldrich Chemical).

The nucleotide sequence of PM77 comprises: 5'-GTA CCG TCA GAA TTC TTC CCT AAG AAA-3'.

The hybrid complex was captured with 50 ug of PMP on which a second binding support partner (avidin) had been immobilized.

The PMP were separated and washed as described in Example 1. In the detection of the captured hybrid complex, the chemiluminescent reaction reagents were modified as described in Examples 1 and 6 in order to obtain maximum sensitivity.

Results were expressed as RLU experimental/RLU background, where RLU background was the chemiluminescent reaction observed in the absence of the added Campylobacter rRNA, and indicate that approximately 100 amol of Campylobacter rRNA was detected, see Figure 2.

EXAMPLE 3 Varying amounts of Campylobacter rRNA (100 amol-100 fmol) were hybridized at 65 0 C for 1.0 hr. with 1.0 pmol labelled oligonucleotide probe (DNP-PM238), the DNP serving as a first support binding partner, and 0.8 pmol labelled oligonucleotide probe (biotin-PM78), the biotin serving as the second support -27binding molecule, in 500 ul of buffer (same buffer as described in Example Following hybridization, the hybrids were either captured on the PMP first and then labelled with a labelled chemiluminescent molecules (avidin-AE), forward format, or labelled first with a labelled chemiluminescent material (avidin-AE) and then captured on the PMP, reverse format. In another variation of the format, the labelled oligonucleotide probe (biotin-PM78), the probe being labelled with the second support binding molecule was prelabelled with the labelled chemiluminescent molecules (avidin-AE). This adduct, labelled chemiluminescent molecules and labelled oligonucleotide probe, (avidin-AE/biotin-PM78) was hybridized directly with Campylobacter rRNA along with the probe labelled with the first support binding molecule (DNP-PM238).

In the forward format, the hybrid was captured by incubation with 50 ug of labelled solid support (5H1-PMP) for 0.50 hrs. at room temperature (230 The PMP were then separated and washed as described in Example 1 and resuspended in 100 ul of buffer containing approximately 5.6 x 106 RLU of labelled Ie chemiluminescent molecules (avidin-AE). After 2.0 hrs. the PMP S were once again separated, washed, resuspended and the chemiluminescent molecules activated. In the reverse format, approximately 5.0 x 107 RLU of labelled chemiluminescent molecules (avidin-AE) were added to each hybrid solution and incubated for 0.25 hrs. at 650 C. Labelled hybrids were then captured by incubation with 50 ug of labelled PMP (5H1-PMP) for -28- 0.50 hrs. at room temperature (230 Samples were processed and the chemiluminescent molecule activated as in the first and second examples. Results of these assays were expressed as signal/background for each amount of rRNA, where background was the signal observed in the absence of rRNA as shown in Figure 3.

EXAMPLE 4 Sandwich hybrids comprised of Campylobacter jejuni rRNA and two derivatized and labelled oligonucleotide probes, one for capture and one for detection, were prepared by combining a various amount of Campylobacter rRNA in 100 ul of 60 mM sodium citrate, 10 mM Tris, 0.6 M sodium chloride 50 mM EDTA and 0.05% SDS (hybridization buffer) with 160 fmol of double-labelled probe 3'- 32 P-PM238) for detection and either 2.0 pmol of an oligonucleotide probe ar Iled with a first support binding molecule (5'-DNP-PM78) or 2.0 pmol of a probe labelled with a second support bindin molecule (5'-biotin-PM78) for capture, and then incubating at 560 C for 2.0 hrs. Following hybridization, 10 ul aliquots were added to 50 ug of capture probe specific solid support (PMP) in 90 ul of 60 mM sodium citrate, 16 mM sodium phosphate, 0.72 M sodium chloride, 0.08% BSA, 0.02% Tween-20, and 0.04% sodium azide at pH 7.2 (phosphate assay buffer). Capture specificity was determined by the binding molecule covalently attached to the labelled PMP, 5H1 anti-DNP-PMP or avidin-PMP to capture sandwich hybrids containing labelled oligomeric probe (DNP-PM78 or biotin-PM78) respectively. Nonspecific binding of the detector molecules to -29the solid support (PMP) was assessed in parallel reactions where excess beta-analine-DNP or biotin, respectively, was added to the PMP to block all binding sites prior to the addition of the sandwich hybrid. After incubation for 0.50 hrs. at room temperature (230 C) the bound sandwich hybrid was isolated from excess unhybridized probes and test sample by separation as described in Example 1. The PMP were washed twice with phosphate buffer and resuspended in 100 ul of distilled water.

Bound sandwich hybrid was detected by chemiluminescent reaction using standard MagicR Lite reagents and liquid scintillation counting. The results of these assays are summarized in Table EXAMPLE The sandwich hybrids described in Example 4 (not bound to PMP) were analyzed by chromotography on a Sepharose CL-6B (Pharmacia) column (Pasteur pipet) which was equilibrated with a buffer containing 0.1 M NaCl, 10.0 mM Tris, 1.0 mM EDTA, pH Oligonucleotide probes hybridized to Campylobacter rRNA S were shown to elute in the void volume and unhybridized oligonucleotide probes were retained in the column. The column fractions were first analyzed by chemiluminescent reaction and subsequently by liquid scintillation counting. The results of S these assays are summarized in Table 6.

The data indicate that the extent of hybridization when assessed by either chemiluminescent reaction or liquid scintillation counting are similar. In contrast to Example 4 where the sensitivity for detection of sandwich hybrid captured upon the PMP was significantly better by liquid scintillation counting as opposed to chemiluminescent reaction, when analyzed by chromotography, the sensitivities of the two detection methods were shown to be similar or slightly better for the chemiluminescent reaction.

EXAMPLE 6 A sandwich hybrid was formed with Campylobacter rRNA by combining 0.5 pmol Campylobacter rRNA, 2.0 pmol labelled oligonucleotide probe (biotin-PM78) and 1.0 pmol labelled oligonucleotide probe (5'-AE-PM238) in 100 ul of hybridization buffer and incubating at 560 C for 2.0 hrs. The resultant hybrid complex and a labelled PMP (avidin-PMP) were mixed, at a ratio of 5.0 fmol of hybrid complex to 250 ug of labelled PMP, and incubated at room temperature (230 C) for 0.50 hrs. to capture the hybrids on the solid support. Bound sandwich hybrid was isolated from excess unhybridized probes and test sample as described in Example 1. The PMP were washed twice with phosphate assay buffer and resuspended in 100 ul of distilled water. Solutions containing the chemiluminescent molecules the labelled oligomeric DNA probe (AE-PM238) and the sandwich hybrid were suspended in distilled water and divided into 100 ul aliquots.

The following parameters associated with the chemiluminescent reaction were examined for purposes of enhancing the detection efficiency of the hybridization assay as -31described in Examples 4 and 5. First, the time-dependence of the chemiluminescent reaction for the above-described solutions was determined under standard MLA conditions by monitoring the output of the photomultiplier tube of the MLA instrument. In each of the samples the activity peaked within one second of the initiation of the chemiluminescent reaction and then subsided to background level readings within two seconds; thus showing that all of the available light signal was being recorded by the MLA during the standard integration.

Since the first step in the chemiluminescent reaction involves attack by hydroperoxide ions on the chemiluminescent molecules (AE) to form an electronically excited molecule, which occurs following addition of reaction reagent 1, the effect of increasing the incubation period of the immobilized sandwich hybrid in the reaction reagent was determined by varying the delay between injection of the reaction reagents in the MLA sequence from the standard value of 0.1 seconds to 10 min. The results of these assays are summarized in Table 7, showing only negligible increases in the signal for a change in unit time.

A matrix of reaction reagents with component concentrations equal to or ten (10) times that of standard reagents were tested for their ability to elicit the chemiluminescent reaction of the AE labelled oligonucleotide probe solutions. All of the modified reaction reagents tested resulted in increased chemiluminescent activity, but the maximum signal enhancement occurred when the acid and base normalities were increased -32ten-fo.d (modified reagents) and the hydrogen peroxide and surfactant concentrations were unchanged relative to standard reaction reagents, Under these conditions the chemiluminescent signal detected by the photomultiplier tube from sandwich hybrid in solution increased 2-fold relative to that observed with standard reaction reagents, and an increase of 13-fold was observed for a captured sandwich hybrid. The results of these assays, summarized in Table 8, have revealed conditions that elicit a chemiluminescent response from an AE-oligomer sandwich hybrid bound to a paramagnetic particle comparable to that observed for the same complex in solution.

EXAMPLE 7 Aliquots of the double-labelled sandwich hybrids formed in Example 4 with biotin-PM78, were captured and processed as previously described except that modified reagents as described in Example 6 were used in the MLA instrument for detection of the chemiluminescent reaction. Following the chemiluminescent reaction, the samples were detected by liquid scintillation counting as described above. The results of these assays are summarized in Table 9; and show that the detection of the S chemiluminescent reaction by the modified reagents was enhanced substantially when compared to the standard reagents, while detection by liquid scintillation counting remained relatively unchanged.

-33- EXAMPLE 8 A sandwich hybrid was formed with Campylobacter rRNA by c- oining the following: 1.0 pmol Campylobacter rRNA, 1.0 pmol labelled oligonucleotide probe (biotin-PM78) and 2.0 pmol labelled oligonucleotide probe (DNP-PM238) in 100 ul of hybridization buffer and incubating at 650 C for 1.0 hr.

Dilutions of this sandwich hybrid were prepared at final concentrations of 2.2 x 10 1 1 M to 2.2 x 10 13 M with 60 mM sodium citrate, 10.0 mM Tris, 1.0 mM EDTA, 0.6 M sodium chloride, 0.1% BSA, 0.01% sodium azide and 0.02% Tween-20 at pH 7.4 (Tris assay buffer) with SDS at 0.05%. Samples (0.45 ml) of the diluted hybrids, 10-14 to 10 16 moles were combined with ug of labelled PMP (5H1-PMP) in 50 ml of Tris assay buffer.

Nonspecific binding to the PMP was assessed in parallel reactions where excess beta-alanine-DNP was added to the PMP to block all binding sites prior to the addition of the sandwich hybrid. After incubation for 0.50 hrs. at room temperature (23 0 C) bound sandwich hybr.d was isolated from excess unhybridized oligonucleotide probes and test sample by S separation as described above. The PMP were washed once in Tris assay buffer and then the immobilized sandwich hybrids were labelled by resuspending the PMP in 100 ul of Tris assay buffer.

containing 6.6 x 106 RLU of chemiluminescent molecule conjugated second binding partner (avidin-AE) and incubating at room temperature (230 C) for 2.0 hrs. The PMP were washed twice in Tris assay buffer to remove unbound avidin-AE and then -34resuspended in 100 ul of distilled water. Replicate samples were processed in an MLA instrument with standard and modified reagents. The results of these assays are summarized in Table The combir.sed results of Examples 4 and 5 suggest that F,, support bound sandwich containing a chemiluminescent labelled oligonucleotide probe is detected less efficiently than the same hybrid in solution when standard reaction reagents are utilized, i.e. only a fraction of the total hybridized chemiluminescent labelled oligonucleotide captured on the solid support were activated by the standard reaction reagents. In Example 4, the detection by liquid scintillation counting resulted in a significantly improved sensitivity, arbitrarily defined as the hybrid input required to generate a S/B value of 2, relative to the detection by chemiluminescent reaction with standard reaction reagents. However, in Example 5, when the same hybrids were analyzed following chromatographic elution, which achieves a solution phase separation of hybridized and unhybridized probes, sensitivity was comparable by both detection methods.

In the first step of the chemiluminescent reaction sequence S a quantity of reaction reagent 1 is injected over a short time period (1.2 seconds the hydrogen peroxide contained in the solution reacts with the chemiluminescent molecules (AE) to form an electronically excited molecule (N-methylacridone). Then following a brief pause, (0.1 second) an equal quantity of a second reagent is injected over a short time interval (1.2 seconds); as the pH becomes basic the excited molecule undergoes and irreversible reaction involving the emission of light. The emitted light signal is detected by a photomultiplier tube which is in physical proximity to sample, and the MLA instrument microprocessor integrates the photomultiplier tube output for a period of time (2.0 seconds), beginning at the addition of the second reagent. The results of Example 6 show that the chemiluminescent signal decreases to background within two seconds demonstrating that all the available signal has been recorded. This applies to the chemiluminescent reactions of the chemiluminescent molecules alone, chemiluminescent molecules covalently attached to oligonucleotide probes, chemiluminescent oligonucleotide probe labelled sandwich hybrid in solution, and solid support bound chemiluminescent oligonucleotide probe labelled sandwich. Consequently, the low efficiency of the detection of solid pha.e bound chemiluminescent oligonucleotide probe labelled sandwich hybrid observed with standard reaction reagents was not due to a delayed activation of the chemiluminescent molecules (AE) in the complex.

In an attempt to increase the amount of excited molecule (N-methylacridone) generated from the solid support bound chemiluminescent oligonucleotide probe labelled sandwich hybrid.

S in the first reaction reagent during the MLA sequence, thereby increasing the signal generated at the addition of the second reaction reagent, the incubation time of samples in the first reaction reagent was increased by varying the delay between -36injection of the first and second reaction reagent up to 6000-fold (0.1 seconds to 10 min.). However, only a modest increase in the signal generated was observed, which did not account for the order of magnitude difference in the sensitivity of detection by chemiluminescence versus liquid scintillation counting.

Finally, substantial enhancement of the signal from the solid support bound chemiluminescent oligonucleotide probe labelled rRNA sandwich hybrid was obtained when the normality of the reaction reagents were increased tenfold, i.e. modified reagents.

It is to be understood that various other modifications will be apparent to and can readily be made by those skilled in the art, given the disclosure herein, without departing from the scope and materials of this invention. It is not, however, intended that the scope of the claims appended hereto be limited to the description as set forth herein, but rather that the claims be construed as encompassing all features of patentable novelty which reside in the present invention, including all features which would be treated as equivalents thereof by those

I..

skilled in the art to which the invention pertains. It is also oir noted that the examples given herein are intended to illustrate, and not to limit the invention.

0 -37- TABLE 1 Base Length PM Location Corresponding to E. Coli 16S rRNA Sequence 74 1.6S-0163-0214 16S-0176-0205 76 16S-0163-0204 77 16S-0437-0463 78 16S-0641-0671 79 16S-0821-0845 16S-0195-0215 122 16S-0641-0671 138 16S-0641-0671 145 16S-0156-0185 154 16S-0829-0854 155 16S-1107-1140 AAC TTT TGT GTT AGA GTA TAC TCA CAG GAG GTT AAG GTA TTA GTA CCG CCT AAG TCT GCC TAG ACT ACT AGC AGT GTA AAC TTT

GT

TCT GCC TAG ATT TCT GCC TAG ATT GGA GTA AGT CAT ACT GCC GCA ACA TGT TAG TGG GTT

CCC

AAG

TTA

ACT

TAT

CAG

GCA

TCA

AAA

TCT

ATG

ATC

C

CCC

TCT

ATC

TCT

ATG

TGG

TTC

GTG

AC

CAA

GCG

TAC

CAG

GCA

TGT

AGA

GAG

GTC

GAA

CCC

AGT

CCA

TAC

CCC

AGT

CCC

AGT

AGT

TAA

ACT

CTA

TCA

GAG

GTC

GTT

TAT

G

TTC

TCA

T

ACA

TCA

TCA

T

TCA

T

ATT

AGC

AAT

ACT

TAT

G

AAG

AGA

TTC

CTC

ACT

ACT

CTC

CTC

AGC

ACA

ACG

238 16S-0706-0732 GCC TTC GCA ATG GGT ATT CTT GGT GAT -38- TABLE 2 (1 of 3) RNA PURIFICATION PROCEDURE 1. Add 0.1 grams wet weight of cells to a bead beater tube.a 2. Discard supernatant. Resuspended cells in 700 ul of buffer 1 by vortexing.

3. Add 50 ul of 20% SDS and 0.1-0.15 mm glass beads to approximately one quarter volume of tube.

4. Add equilibrated phenol to fill tube.b Cap and beat for four minutes at room temperature in bead beater.

6. Submerge tubes in water bath (60 0 C) for fifteen minutes to deproteinate and break up DNA.

7. Dry outside of tube and beat for an additional two minutes.

8. Spin at 3000 RPM (735 x for five minutes in a microfuge. DO NOT USE MICROFUGE AT HIGH SPEEDS! Alternately, spin in the speed vac with no vacuum for ten minutes.

9. Remove nearly all of the aqueous phase (top layer) and the interface to a fresh sterile 1.5 ml microfuge tube leave the beads and the phenol behind).

Add phenol to fill, and then vortex for 1.5 minutes.

11. Microfuge for 5 minutes at 8,000 RPM (5220 x Remove S aqueous phase to new tube taking a small amount of phenol if necessary.

12. Phenol extract (steps 10 and 11) twice more.

-39- TABLE 2 (2 of 3) 13. Phenol/chloroform (Phenol/chloroform/isoamyl alcohol 2 5 2 4 :1 c extract twice as in steps 10 and 11 except be sure to leave all interface and bottom layer behind after the last extraction.

14. To remaining aqueous phase add 0.1X volume of 3M sodium acetate, pH 5.2.

Aliquot in tubes (50 ul per tube).

16. Add two volumes of cold ethanol. Vortex briefly.

Precipitate at -70 0 C overnight. Long term storage of the prep should be in this form and at this temperature.

17. To one of the ethanol precipitate tubes, microfuge at 14,000 RPM (16000 x for ten minutes. Decant ethanol and dry pellets in speed vac (usually five minutes under vacuum is sufficient).

18. To determine optical density resuspend dried pellet in 300 ul of DEPC-treated water. Dilute this sample 1:10 in DEPC water and measure absorbenciss at 260 and 280 nm. Discard, the solution that went into the cuvette and store the remainder at pop -70 0 C. This is the working solution.

)*L

a. To prepare cells, grow confluent lawn of bacteria on plates. Each plate generally yields 0.1-0.2 g cells, depending on strain.

1) "Shave" the plates with a sterile razor blade and transfer the cells to a pre-weighed 50 ml plastic conical centrifuge tube.

TABLE 2 (3 OF 3) 2) Add approximately ten ml LB medium, vortex to resuspend the cells.

3) Spin in Beckman TJ-6R table top centrifuge for minutes at 2400 RPM in TH-4 rotor (1200 x 4) Remove supernatant.

Weigh the tube and cells to determine wet cell weight.

6) Add 1 ml LB medium per gram of wet cells.

7) Transfer 0.1 ml wet cells 0.1 g) to bead beater tube for RNA preparation. Spin 5 minutes at 3,000 RPM (735 x in microfuge. Discard supernatant. Proceed with this sample as described in main protocol, above.

8) Spin the remainder in table top centrifuge for minutes at 2400 RPM in TH-4 rotor (1200 x 9) Remove the liquid. Store cells at -70 0

C.

As an alternative method to harvest the bacteria: 1) Add 1 to 2 ml LB to each plate. Resuspend cells in LB with pipetting action or by dislodging cells with a curved glass rod.

2) Transfer cells to pre-weighed 50 ml plastic conical centrifuge tube. Fill to 10 ml with LB medium.

3) Perform steps 3 to 9 as described above.

-41b. To equilibrate phenol, add equal volume of desired buffer, mix vigorously for about 30 seconds, and separate layers by spinning 5 minutes at 500 RPM in TH-4 rotor (50 x in the table-top centrifuge. For twice equilibrated phenol, remove the aqueous phase from the equilibrated phenol and repeat.

c. For phenol/chloroform/isoamyl alcohol, mix equal volumes of equilibrated phenol and chloroform/isoamyl alcohol premixed at a ratio of 24:1.

Il 08

TL

-42- TABLE 3 (1 of P-.omega lorganism name Gel 28 1018 Klebsiella pneumoniae 36,25 i'e 107A Staphylococcus aureus 258 28 1038 Salmonella enter iditis 25 PB 10 4A1 Escherichla colt 1 PB 104AZ Kscherichia colt 19 P8 105B Proteus .2irabills 25 PB 106AI Streptococcus Group D 1 P8 106A2 Streptococcus Group D 19 PB 107B Serratla marcescens 36 PB 1088 Pseudomonas aeroqictosa I PB 1098 Edvardslella tarda bq 1 1 PB 110A Enterobacter agglomerans 13 PB 111A Shigella sonnet I PS 1128 Yersilia enterocolitica 36 PB 1138 Aclnetobacter calcoacetic36 PB 114 Campylobacter Jejuni PB 115,X Camplobacter jejuni 459 PS 116A Caripylobacter Jejuni 1 PR 117 Campylobacter jejuni PB 118A Canpylobacter colt 1 PB 1188 Camnylobacter colt 18 PB 119A CaMplobacter fetus 29 P8 120A Campylobacter jejuni 1 PE 121A Aeromonas hydrophila. 25 PS 122A Aeromonas sorbia I PB 123A Citrobacter freundil 1 PB 124A Citrobacter freundil 1 PB 125A Klebsiella. pneumoniae 1 PB 126k Enterobacter cloacae 1 PB 121A Pleisiomonas shigelloidesol,49 P981278 Pleisiomonas shigelloidesi PB 128A Salmonella arizonae 36 PB 129A Salmornella typhimurium 41,49 PB 130A Shigella sonnet 1 PS 131A Vibria parahemolyticus PS 132A Streptococcus agalactiae PS 133A Streptococcus faecalis 47,47 PB81338 Streptococcus faecalis 1 PB 134k Streptococcus group C 47,47 PS 1348 Streptococcus group C 18 PS 135A Campylobacter jejuni 26 PS 136A Campylobacter fetus 28 P9 137A Campylobacter lanidis 1 PS 138A Morganella morganil 47,47 28 1388 Morganella rmorganii 1 PB 139A Proteus mirabilis 36,36 PS 140A Providencia stuartil 28 PB 201,kl Campylobacter jejuni 1 PO 20lA2 Campylobacter jejuni 1 PB 202A Campylobacter jejuni 1 PB 203AI Campylobacter jejuni 1 PB 203A2 Caapyo,cter jejuni 19 PS 203)L3 Campylobacter jejuni 19 PB 20381 Campylobacter jejoni 19 PS 20382 Campylabacter jejuni 19 PS 203C Campylobacter jejuni 19 P9 204A Campylobacter jejuni I PO 205A Campylobacter jejuni 25 PO 206A Campylobacter colt 28 PS 207A (.amnv~nrirt.r ipiiint IR 74 76 78 80 138 154 231 0 0 0a0 79 122 145 155 0 00 0 0 00 0 0 0 0 00 0 0 0 0 00 0 0 0 00 0 0 0 0 00 0 0 0 0 00 a0 0 a0 0 a00 00 0 0 0 00 0 0 0 00 0 0 0 0 0 00 0 0 00 0 2 2 2 2 22 2 2 2 2 2 2 2 22 0 t 0 2 2 22 2 0 0 00 0 00 0 0 00 0 0 0 00 0 00 0 0 0a0 2 2- 2 2 02 2 200 a22 0 0 00 0 00 0 0 0 0 0 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 0 00 0 0 22 2 22 2 22 2 22 2 22 2 22 2 2 2 0 0 2 0 20 0 200 2 0 2 0 2 0 0 *0 u -43- TABLE 3 (2 of Prozeqa 8Organism name IPB 208A Campylobacter jejuni PS 209A Campylobacter jejuni PIS 210A Campylobacter jejuni P8 211A Campylobacter jejuni PS 212A Caumpylobacter jejuni P8 213A Campylobacter jejoni P8 213B Campyobacter Jjini PB 213C Catpylobacter ieiudi PB 214A Carpyobacter jejuni P8 2148 Caapylobacter jejoni PB 215A Campylobacter colt PB 216A Campylobacter jejuni PB 217A Campylobacter jejuni PB 218A Campylobacter je~uni PS 219A Campylobactet jejuni PB 220h Campylobactet jeJuni PB 221A Campylobacter colt PB 222A Campylobacter jejuni PB 21 Campylobacter jejuni ?a 224A Campyiobacter jejun! P3 225A Camoylobacter colt P3 226A Campylobacter coli PB 227A Campylobacter colt PB 228K Camoylobacter jej~nni P3 228B Campyiobacter jejuii ?B 229K Camylobacter coil PB 229B Campylobacter colt P3 230A Campylobacter jejuLni PB 2303 Campylobacter jejuni ?S 231A Campylobacter colt PB 232A Campylobacter jejuni PB 2325 Campylobacter jejuni P3 233A Campylobacter jejuni PB 234A Campylobacter Jejuni PB 235A Campylobacter colt PB 236A Camylobacter jejuni PB 236B Campyobacter jejuni PB 237A Campylobacter jejuni PB 238K Campylobacter jejoni 93 B8 Campylobacter jejun! PB 239A Campylobacter jejoni PB3 240K Campylobacter colt PB 301A Campylobacter jejuni PB 302A CaM~ylobacter jejuni PB 3031 Campylobacter jejoni PB 304A Campylobacter jejuni PB 305K Campylobacter jejuni PB 305B Campylobacter jejuni PB 306A Campylobacter jejuni PB 307A Campylobacter coli PB 308K Campylobacter colt PB 309A Campylobacter colt PB 310A Campylobactet colt PB 311A Campylobacter jejuni PB 311B Campylobacter jejuni PB 312K Campylobacter colt PB 312B Campylobacter colt PB 313K Campylobacter jejuni PB 3138 Campylobacter jejuni PB 314K Campyobacter jejufli Gel1 28 1 79 17 1 1 19 48 49 258 18 28 48 18 48 18 18 49 1 49 48 47 458 47 458 23 47 458 458 458 358 37 1 49 46 1 49 49 36 468 468 46 37 1 469 18 is 1 18 47 48 46 48 47 48 Al 74 76 78 80 138 154 238 77 79 122 145 155 22 22 2 22 2 02 2 22 22 2 22 02 2 2 2 22 2 02 2 22 22 22 2 22 02 2 22 22 2 22 2 02 2 2 22 2 2 22 22 2 2 2 22 2 2 22 2 2 22 2 22 22 2 22 2 22 2 22 2 22 2 2 2 2 02 1 2 22 2 2 22 2 22 22 22 2 2 02 2 2 0 2 2 0 2 2 0 2 2 0 2 2 8 2 2 0 2 2 02 2 1 02 2 02 2 0 22 0 22 0 22 0 22 0 22 0 22 0 22 0 22 -44- TABLE 3 (3 of Promega lorganisa name Gel PB 314a Campylobacter jejuni 48 PB 315?. Campylobacter coli 49 PB 316GA Campylobacter Jejuni 46 PB 316B Campylobacter jejuni i8 PB 317A. Campylobacter coil 38 PB 318A Campylobacter jejuni 47 PS 3188 Campylobacter jejuni 48 PB 319A. campylobacter jejuni 47 PB 3198 Campylobacter jejuni 48 PB 320A Campylobacter coll 47 PB 320B Campylobacter coli 18 PB 321A. Campylobacter jejuni 47 PB 3218 Campylobacter jejuni 48 ^B 322?. Tersija enterocolitica 49 PB 323?. Iterobacter aerogenes 25 PB 324A. Enterobacter cloacae 1,1.

PB 325A. Morganella morganli 1 PS 326A Providencia rettqeri I PB 327A. Iinetobacter calcoacetic48 PB 328A. Escherichia cmli A-0 47 PB 329A. Salmonella st paul.

PB 330A. Salmonella give 28 PB 331A. Salmonella mississippi 28 PS 332A Shigella boydi! I P3 333A Shigella dysenteriae 25 PS 334A Shigella. flexneri I PS 335A Streptococcus agalactiae 47 PS 335B Streptococcus agalactiae I P9 336GA Candida. albicans 25 PS 337A Candida tropicalis 36 P3 338A Staphylococcus aureos 258 P8 339A. Staphylococcus epideraidil9 PB 340k Pravidencia stuartil 28 PS 401k Salmonella schvarzengrundl8 PS 402A Salmonella typhi PB 403AI, Volinella species I HB 403A2? lolinella species 36 PB 403A3 Volinella species 25 PS 404A Volinella curva 479 PB 40481 *olinella curva 48 PS 40482 Volinella curva 48 P8 40483 Volinella curva, 36 PB 405A. Volinella recta 48 PS 40GMI Volinella succinogenes 1 *PB 406A2 Volinella succinogenes 25 PB 406k3 Tolinella succiaoqenes 25 PB 407?. Campylobacter hyointestinl P848A Campylobacter cinaedi 47 P* P408B Campylobacter cinaedl 479 PB 408C Campylobacter ctnaedL 419 SPS 408D Campylobacter cinaedi 479 PB P409A Escbericbia, coli 0157:H17 I PB 410k Escherichia coil 0157:H7 1 PB 411,U Vibrio cholerae 1,1 PS 411A2 Vibria cholerae 1 iP8 412A ViIrio choierae 47,1 I28 413A Bact. fragilis 1 PB 114A1 Bact. fraqilis I P8' 4IIA2 Bact. fragilis 25 74 76 2 2 78 79 2 2 2 2 80 138 154 238 122 145 155 2 02 2 2 02 2 2 2 2 2 2 2 2 02 2 2 2 02 2 2 2 2 2 2 2 02 2 0 22 02 2 2 2 2 2 2 2 0 00 0 0 000a 0 0 00 0 00 0 0 0a0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 00 0 0 00 0 0 00 0 2 2 0 0 0 0 0 0 0 0 0 0 0 0 a 0 0 00 0 0 0 0 0 0 0 0 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00r 0 0 006 0 0 0 0 0 0 0 0 00 0 0 00 0 0 0 2 0 0 0 0 0 2 0 1 0 0 0 00 0 0 0 0 0 0 0 0 08 0 1 0 1 0 1 0 2 2 0 0 0 0 0 0 0022 0 0 0 0 0 0 00a 0 a 0 0 0 0 0 TABLE 3 14-o5T Promega forganisa name Gel PB 415A Bact. thetaiotaxicron 1 PB 416A Bact. vulgatus I PB 417A Clostridium perfringens PB 4178 Clostridiun perfrlngens I PB 418A Clostridlum perfringens 59 PB 419AI Clostridium ramosum 1 PB 419A2 Clostrldium ranosum I PB 42Okl Clostridini sordellil 18 PB 42OA2 CIlostrldlut sordelli 18 PB 420A3 Clostridium sordelll 18 PB 421AI Clostridium septicum 1 PB 421A2 Clostridlum septicum 36 PH 422A Clostridium tetani 47 PB 423A Clostridium, tetani I PB 424k Peptostrep. anaerobius 1 PB 425A Bact. distasonis 36 PB 426A Bact. multiacidus 1 PB 427A Bifidobacterium breve PB 428A Fusobacterium nucleotum PB 428B Fusobacterium nucleotum 26 PB 429A Bact. melantinagenicus 36 PB 430A Camp lobacter upsaliensisi PB 431A Campylobacter upsaliensisj6 PB 432k AnaerobiospirilluR sac. 1 PB 433A Campylobacter fennefliae 479,479 PB 433B Campylobacter fennelliae 479 PB 433C Campylobacter fennelliae 479 PB 434A Campylobacter fennelliae 479,479 PB 434B Campylobacter fennelliae 479 PB 434C Campyiobacter fennelliae 479 PB 435A Eubacterium aerofaciens 36 PB 436k Pentostreptococcus produc36 PB 439k Coprococcus eutactus 1 PB 439B1 Coprococcus eutactus 1 PB 43982 Coprococcus eutactus I PB 440 A CamDlobacter concisus 1 PB 501 A Bact. gracills 46 IPB 501B Bact. gracilis 48 PB 502A Bact. ureolyticus 468 PB 503A Veillonella parvula 1 PB 504A Streptococcus aitis 1 PB 505A Mlcallgenes faecalis 37 PB 5058 Alcaligenes faecalis 1 PB 506k Bacillus cereus 1 PB 507A streptococcus pyogenes 18 PB 508k leisseria gonorrhoeae 1 PB 509k Ca~pylobacter fetus 38 PB 510k Caspylobacter Cali 1 IPB SloB camaylobacter call 19 PB 511A Campylobacter fetus 38 PB 512k Caopylobacter fetus 48 PB 513A Caap lobacter fetus 38 PB 51.4A1 Camnylobacter fetus 38 PB 514A2 Campylobacter fetus 18 PB 515A Campyinbacter fetus 38 PB 516k Cs-pylobacter fetus 38 PB 5 17k Campylobacter fetus 38 PB 518A Canpylobacter laridis 1 PB 519k Campylobacter laridis I PB 5198 Camnylobacter laridis 1 74 76 78 50 138 154 238 77 79 122 145 155 00 0 00 00a0 0 0 00 00 0 00 0 00 0 0 0 0 0 0 0 G0 a0 0 0 0 0 0 0 0 0 0f 0 0 2 0 0 0 0 020 01a I -46- Promega fOrganisa ux Gel 74 76 78 30 138 1,54 238 77 79 122 145 155 1 2 22 2 0 22 ?3 520A Campylobacter larldis I PS 5211 Campylobacter larldis 47 ?9 5218 Camylabacter laridis I 28 5221. Caupylobacter pylori 19 P8 5221.2 Campylobacter pylori 18 PB 523A. Campylobacten pyluri 19 ?B 5241. Capylobacten pyloni 58 PS 5251. Caspylobacten pylorn 18 PS 533A.1 Zscherichla coil 1 PB 533A.2 Zscherichia coil 1 PS 5338 Eschenlchla coil 1s PS 603A. Lactobacillus acidophilusi 604A. Fusobacterica necroohonuolS PS 701A. Clostridlum difticile 258 7hi- 'i r.s o ~urn i!a t e! s I.!in nurr.jr u ;eo At P:-cmega Corpor.ation.

Thasecond gir ~i cn-,c ar spepcies mire for echoreanis:-.. The iC c~iut t.;c ±i,;rp -t&Licn after agavos a g-1el ~ectrophoresis of zr-i:y o~f n rpA -fiied from eaoh. strai1n, T~c Tajor bards were tac fore~:e Sa~ h t .ai wal- pr .surm-,d be 23S arnd the lower ta., r et c 1'0 Ex:.r'a banc, 'ir 3ocme'..Imes observed ;Ind mnbanl s we'e- ssln8g a:cor-ing th- fcllowing k:ey: I abcth arcsw:C:;~r t norn--i th tcp t;.wa stron,;1y 'jr, rr2pen.ent?6 9~prp .ir ;ai:5 tWO Cr M.Cre extra bands NWh'r. the 13e!" h:.s r-.:ltiple numtsirs in itall lszt ,ri cases Pply.

n trr; -itt :1 c tatle, (2 ndcPct-:s ro hybriaizat ion, in--'!cate~s ~zzn hbrciz~ic, d ~iviatie3 weak Bliii~l.r.~ ank~ spaces or:c.~ r:c: t. r r- -nt rt a: ion a 1;Ar c u ;.ir pr o w it n a p a r C: u Ia r -47- TABLE 4 CAMPYLOBACTER PROBES HYBRIDIZATION SUMARY 74 75 76 77 78 79 80 122 138 145 154 155 238 C. jejuni, coli, laridis C. fetus C. cinaeai C. hyointestinalis C. pylori C. upsaliensis Bacteroides gracil is Bacteroides ureolyticus Wolinella curva Wolinella recta All others 0 0 0 0 0 0 O 1- ~1o 0 0 0 0 0 0 0 0 0 0 0 0 =NOT D:ETERMINED =Hybr-idization -=No Hybridization et. S..

S S S S 'S S TABLE 5H1 PMP Capture Streptavidin PMP Capture CHEMILUMINESENCE LIQUID SCINTILLATION CHEMILUMINESENCE LIQUID SCINTILATION (Fmol (RLU) 1 (CPM) 1 (RLU) 2 (CPU) 2 rINA Tnput Signal S/B Signal S/B Signal S/B Signal S/B 0.1 1 100

NSB:

0.1 1 100 645 1,595 8,970 14,500 665 635 625 660 l.:J 2.5 14 22 x 645 66 438 3,313 4,947 38 29 38 31 1.9 13 97 145 470 885 4,200 10,050 1.1 2.0 9.4 23 x 445 66 454 3,487 5,909 31 33 38 38 1.9 13 100 169 x x 34 Sensitivity 0.71 Fmols 0.10 Fmols 1.0 Fmols 0,11 Fmols 1: Signal to Background (S/B) is the dividend of the chemiluminescent signa' cbserved (Relative Light Units (RLU)) at a given rRNA input divided by the mean of the non-specificaliy bound chemiluminescent signal observed at all rRNA inputs.

2: Signal to Background is the dividend of the liquid scintillation signal observed (counts of radioactivity) (CPM) at a given rRNA input divided by the mean of the non-specifically bound (NSB) liquid scintillation signal observed at all rRNA inputs.

3: Sensitivity is defined as the amount of sandwich hybrid, in femtomoles (1 X 101-15) required to generate S/B of 2; calculated by linear interpolation.

TABLE 6 (fmoi rRNA Input Hybrid CPM Capture Probe Hybrid RLU Hybridization Hybridization I 'I 4- ii II a' 1 100

DNP-PM

DNP-PM

DNP-PM

DNP-PM

Biotin Biotin- Biotin Blotin 1,200 18,000 140,000 334,000 2,000 22,600 189,000 336,000 750 4,530 8,360 280 5,260 8,900 a a a a TABLE 7 (seconds) Delay Between Inj ections

MRU)

Signal observed with standard Reagents Hybrid In SolutionI PMP-Bound Hybrid ii 150 300 600 *328,000 339,000 349,000 345,000 345,000 350,000 345,000 382,000 2,200 3,350 3,200 3,250 3,100 2,900 2,800 3,000 -4 0** .3 .3 TABLE 8 Magic-Lite Analyzer Reagents

(RLU)

Reagent Background (Moles) Hybrid Input

(RLU)

Solution Chemiltuminesence

(RLU)

PMP- Captured Chemiluxninesence Standaid 370 5 X 10 -15 48,360 j1,980 Modified JJ 950 5 X 10 -15 78,830 124,920 Ratio 1:Ratio is equal to the dividend of the chemiluminescent signal observed upon activation of the MLA system with Modified Reagents divided by that observed upon activation with Standard Reagents.

S C. a TABLE 9 CHEMILUMINESENCE LIQUID SCINTILLATION (fmol) rRNA Input

(RLU)

Signal

(CPM)

Signal S/B 0.1 1 100 NSB: 0.1 1 100 Sensitivity 3 1,530 8,725 72,750 127,250 745 880 800 900 0.1; 1.8 10.5 88 150 76 364 2,970 4,648 2.2 11 87 140 X 34 X 830 Sfmoi ~0.1 fmol 1: Signal to Background is the dividend of the chemiluminescent signal observed on the MLA system at a given rRNA input divided by the mean of the non-specifically bound (NSB) chemiluminescent signal observed at all rRNA inputs.

2: Signal to Background is the dividend of the liquid scintillation signal observed at a givern rRNA input divided by the mean of the non-specifically bound (NSB) liquid scintillation signal observed at all rRNA inputs.

3: Sensitivity is defined as the amount of sandwich hybrid, in femtomoles, required to generate S/B of 2; calculated by linear interpolation.

ft TABLE STANDARD REAGENTS MODIFIED REAGENTS II (final) rRNA Input

(RLU)

Signal

(RLU)

Signal 2

S/B

II I JL 0.1 0.32 1 3.2 0.1 0.32 1 3.2 sensitivity 2 1,795 3,005 4,500 18.,485 50,455 1,030 1,060 1,200 1,025 1,205 1.6 2.7 4.1 17 46 2,445 3,680 7,225 18,885 53,520 1,630 1,685 1,185 1,960 1,285 1.7 13 37 x 1,100 0.18 final X =1,450 0. 17 final 1: Signal to Background is the dividend of the cheinilumnine scent signal observed on the MLA system at a given rRNA input divided by the mean of the non-specif ically bound (NSB) cheiniluiinescent signal observed at all rRNA inputs.

2: Sensitivity is defined as the amount of sandwich hybrid, in femtoinoles, required to generate S/B of 2; calculated by linear interpolation.

Claims (36)

1. A method for assaying Campylobacter rRNA including: a. providing a test sample including cells of one or more cell types, said test sample including cells of one or more cell types, said test sample suspected of containing Campylobacter cells or Campylobacter rRNA, and wherein said Campylobacter cells or Campylobacter rRNA may include one or more species of Campylobacter; b. releasing rRNA from the cells of said test sample; c. hybridizing rRNA of Campvlobacter, if present, in said test sample, with at least two labelled oligonucleotide probes to form a hybrid complex, each of said probes having a nucleotide sequence that is complementary and at least one of which is specific to a region of Campylobacter 16S rRNA, and wherein at least one of said probes is labelled with one or more first support binding partners, and wherein at least one of said probes is labelled with one or more detector molecules; d. capturing said hybrid complex on a solid support ,to form a sandwich complex, said solid support having one or more second support binding padieles immobilized thereon which are complementary to said first support binding partners, and wherein said first support binding partners bind to said second support binding partners; e. isolating said sandwich complex from said test sample and excess nonhybridized probes, f. detecting the presence of Campylobacter by the activation of detector molecules associated with said sandwich complex; and g. quantitating the number of Campylobacter cells in said test sample/anlwherein the sensitivity of said test assay permits quantitation of approximately 1 x bacteria cells per milliliter of test sample.

2. The method as defined in claim 1, wherein said first support binding partner is a hapten, and wherein said second support binding partner is an antibody.

3. The method as defined in claim 1 or 2, wherein said oligonucleotide probes include at least two distinct nucleotide sequences of the group including: a. 5'-AAC TTT CCC TAC TCA ACT TGT GTT AAG CAG GAG TAT AGA GTA TTA GCA GTC or 55 b. 5'-TAC TCA ACT TGT GTT AAG CAG GAG TAT AGA-3', or c. 5'-GTT AAG CAG GAG TAT AGA GTA TTA GCA GTC or d. 5'-GTA CCG TCA GAA TTC TTC CCT AAG AAA-3', or e. 5'-TCT GCC TCT CCC TCA CTC TAG ACT ATG ACT or f. 5'-ACT AGC ATC CCA ACA ACT AGT GTA or g. 5'-AAC TTT CCC TAC TCA ACT TGT-3', or h. 5'-TCT GCC TCT CCC TCA CTC TAG ATT ATC AGT or i. 5'-TCT GCC TCT CCC TCA CTC TAG ATT ATG AGT or j. 5'-GGA GTA TGG AGT ATT AGC AGT CAT TTC TAA-3', or k. 5'-ACT GCC GTG ACT AGC ACA GCA ACA AC-3', or I. 5'-TGT TAG CAA CTA AAT ACG TGG GTT GCG-3'.

4. The method as defined in any one of claims 1 to 3, wherein said detector molecule is a chemiluminescent molecule. The method as defined in claim 4, wherein said chemiluminescent molecule includes an acridinium ester.

6. The method as defined in claim 4, wherein said chemiluminescent molecule includes a dimethylacridinium ester.

7. The method as defined in any one of claims 1 to 6, wherein said solid support includes paramagnetic particles.

8. The method as defined in any one of claims 1 to 7, wherein said detector molecule is activated by the addition of a first reagent solution and then following an incubation time the addition of a second reagent solution, so that a detectable light reaction is uninitiated, and wherein said first reagent solution includes 1.0 N HNO 3 in a solution of H 2 0 2 and wherein said second reagent solution includes 2.5 N NaOH in a 0.5% solution of a surfactant.

9. The method as defined in claim 8, wherein said surfactant is Arquad.

10. The method as defined in claim 1, wherein said first support binding partner S includes avidin/strepavidin or biotin, and wherein said second support binding partner includes buotin or avidin/strepavidin respectively.

11. The method as defined in claim 2, wherein said first support binding partner includes dinitrophenol, and wherein said second support binding partner includes anti- dinitrophenol antibodies. 56 170J2. The method as defined in claim 1, whereinsaid detector molecule is an enzyme.

13. The method as defined in claim 1, wherein said first support binding partner is an antibody or antigen, and wherein said second support binding partner is an antigen or antibody respectively.

14. The method as defined in claim 1, wherein said rRNA is released from cells not in the presence of a stool, by lysis in 0.25% SDS in Tris, EDTA buffer (pH 8-9) for minutes at room temperature, and wherein the release of rRNA from cells in the presence of stool is by lysis in 0.25% SDS in Tris, EDTA buffer (pH 8-9) for 10 minutes at 750C and then filtered. A test kit suitable for detecting and quantitating Campylobacter in a test sample including: a. a solution for releasing rRNA from Campylobacter cells; b. a first labelled oligonucleotide probe, said probe being complementary to Campylobacter rRNA, and wherein said label is a first support binding partner; c. a second labelled oligonucleotide probe, said probe being complementary to Campylobacter rRNA, wherein said label is a detection molecule; and wherein said first probe or said second probe is specific to a region of Campylobacter 16S rRNA; d. a solid support, said solid support having a second support binding partner bound thereto which is complementary to said first support binding partner.

16. An oligonucleotide probe which is complementary and specific for Campylobacter 16S rRNA, said probe having a nucleotide sequence including: CCG TCA GAA TTC TTC CCT AAG AAA-3'.

17. An oligonucleotide probe which is complementary and specific for Campylobacter 16S rRNA, said probe having a nucleotide sequence including: 5'-TCT GCC TCT CCC TCA CTC TAG ACT ATG AGT T-3'. 8. An oligonucleotide probe which is complementary and specific for S. Campylobacter 16S rRNA, said probe consisting a nucleotide sequence of the group including: a. 5'-AAC TTT CCC TAC TCA ACT TGT GTT AAG CAG GAG TAT AGA GTA TTA GCA GTC or b. 5'-TAC TCA ACT TGT GTT AAG CAG GAG TAT AGA-3', or c. 5'-GTT AAG CAG GAG TAT AGA GTA TTA GCA GTC or d. 5'-GTA CCG TCA GAA TTC TTC CCT AAG AAA-3', or ("-0I~k .c3/z II,~ 57 e. 5'-TCT GCC TCT CCC TCA CTC TAG ACT ATG ACT or f. 5'-ACT AGC ATC CCA ACA ACT AGT GTA or g. 5'-AAC TTT CCC TAC TCA ACT TGT-3', or h. 5'-TCT GCC TCT CCC TCA CTC TAG ATT ATC AGT or i. 5'-TCT GCC TCT CCC TCA CTC TAG ATT ATG AGT or j. 5'-GGA GTA TGG AGT ATT AGC AGT CAT TTC TAA-3', or k. 5'-ACT GCC GTG ACT AGC ACA GCA ACA AC-3', or 1. 5'-TGT TAG CAA CTA AAT ACG TGG GTT GCG-3', or m. 5'-GCC TTC GCA ATG GGT ATT CTT GGT GAT-3'.

19. A method for assaying the presence of one or more Campylobacter rRNA sequences in a test sample including: a. providing a test sample including cells of one or more cell types, said test sample suspected of containing one or more Campylobacter rRNA sequences; b. releasing rRNA sequences from "ie cells of said test sample; c. hybridizing the rRNA of Campylobacter sequence, if present in said test sample, with a plurality of distinct oligonucleotide probe units to form a plurality of hybrid complexes, each of said probe units including at least two labelled oligonucleotide probes, each being complementary to and at least one of which is specific to a region of the Campylobacter rRNA and wherein at least one of the probes of said unit is labelled with one or more first support binding partners, and wherein at least one of the probes of said unit is labelled with one or more distinct detector molecules; and wherein said distinct detector molecules are activated sequentially or simultaneously; d. capturing said hybrid complexes on a solid support to form sandwich complexes, said solid support having one or more second support binding partners immobilized thereon which are complementary to said first support binding partners, and wherein said first support binding partners bind to said second support binding partners; e. isolating said sandwich complexes from said test sample and nonhybridized probes of said units; f. detecting the presence of one or more Campylobacter rRNA sequences by the activation of the detector molecules associated with said sandwich complexes, S/ and wherein each of said detector molecules provides a discernible activation 58 reaction; and g. quantitating the number of Campylobacter cells of one or more genus or species in said test sample, and wherein the test assay permits quantitation of approximately 1 x 104 bacteria cells per milliliter of test sample.

20. The method as defined in claim 19, wherein said first support binding partner is a hapten, and wherein said second support binding partner is an antibody.

21. The method as defined in claim 19 or 20, wherein said detector molecule is a chemiluminescent molecule.

22. The method as defined in claim 21, wherein said chemiluminescent molecule includes a dimethylacridinium ester.

23. The method as defined in any one of claims 19 to 22, wherein said solid support includes paramagnetic particles.

24. The method as defined in any one of claims 19 to 23, wherein said chemiluminescent molecule is activated by the addition of a first reagent solution and then following an incubation time the addition of a second reagent solution, so that a detectable light reaction is initiated, and wherein said first reagent solution includes N HN03 in a 0.5% solution of H 2 0 2 and wherein said second reagent solution includes N NaOH in a 0.5% solution of a surfactant. The method as defined in claim 24, wherein said surfactant is Arquad.

26. The method as defined in claim 21, wherein said chemiluminescent molecule includes an acridinium ester,.

27. The method as defined in claim 19, wherein said first support binding partner includes biotin or avidin/strepavidin and wherein said second support binding partner includes biotin or avidin/strepavidin respectively.

28. The method as defined in claim 20, wherein said first support binding partner includes dinitrophenol, and wherein said second support binding partner includes anti- dinitrophenol antibodies.

29. The method as defined in claim 19, wherein said detector molecule is an enzyme.

30. The method as defined in any one of claims 19 to 29, wherein said target nucleic acid sequences include the 5S or 16S or 23S subunit of ribosomes of a genus or species of bacteria. ,i31. The method as defined in claim 19, wherein one of said units includes at least -59 two members of the group including the nucleotide sequences: a. 5'-AAC TTT CCC TAC TCA ACT TGT GTT AAG CAG GAG TAT AGA GTA TTA GCA GTC cr b. 5'-TAC TCA ACT TGT GTT AAG CAG GAG TAT AGA-3', or c. 5'-GTT AAG CAG GAG TAT AGA GTA TTA GCA GTC or d. 5'-GTA CCG TCA GAA TTC TTC CCT AAG AAA-3', or e. 5'-TCT GCC TCT CCC TCA CTC TAG ACT ATG ACT or f. 5'-ACT AGC ATC CCA ACA ACT AGT GTA or g. 5'-AAC TTT CCC TAC TCA ACT TGT-3', or h. 5'-TCT GCC TCT CCC TCA CTC TAG ATT ATC AGT or i. 5'-TCT GCC TCT CCC TCA CTC TAG ATT ATG AGT or j. 5'-GGA GTA TGG AGT ATT AGC AGT CAT TTC TAA-3', or k. 5'-ACT GCC GTG ACT AGC ACA GCA ACA AC-3', or I. 5'-TGT TAG CAA CTA AAT ACG TGG GTT GCG-3', or m. 5'-GCC TTC GCA ATG GGT ATT CTT GGT GAT-3', and wherein said nucleotide sequences are specific and complementary to Campylobacter 16S rRNA, and wherein Campylobacter cells are suspected of being present in the test sample.

32. The method as defined in claim 19, wherein rRNA is released from said cells, not in the presence of stool, by lysis in 0.25% SDS in Tris, EDTA buffer (pH 8-9) for minutes at room temperature, and wherein the release of rRNA from cells in the presence of stool by lysis in 0.25% SDS in Tris, EDTA buffer (pH 8-9) for 10 minuted at and then filtered.

33. A test kit suitable for detecting and quantitating the presence of one or more Campylobacter rRNA sequences in a test sample including: a. a solution for releasing rRNA sequences from the cells; b. at least two distinct first labelled oligonucleotide probes, each of said probes being complementary to a Campylobacter rRNA sequence and wherein said label is a first support binding partner; c. at least two distinct second labelled oligonucleotide probes, each of said probes having a discernible label and being complementary to a target nucleic acid sequence and wherein each of said probes being complementary to a Campylobacter rRNA sequence as is one of the first probes, and wherein said S' label is a detector molecule and wherein at least one of either said first probes 60 or said second probes, complementary to the same target nucleic acid sequence is specific to a region of the Campylobacter rRNA sequence; and d. a labelled solid support, and wherein said label is a second support binding partner which is complementary to said first support binding partner.

34. The method as defined in claim 1, wherein said oligonucleotide probes correspond to the region including: a. bases 0163-0214 of E. Coli 16S rRNA, or b. bases 0176-0205 of E. Coli 16S rRNA, or c. bases 0163-0204 of E. Coli 16S rRNA, or d. bases 0437-0463 of E. Coli 16S rRNA, or e. bases 0641-0671 of E. Coli 16S rRNA, or f. bases 0821-0845 of E. Coli 16S rRNA, or g. bases 0195-0215 of E. Coli 16S rRNA, or h. bases 0156-0185 of E. Coli 16S rRNA, or i. bases 0829-0854 of E. Coli 16S rRNA, or j. bases 1107-1140 of E. Coli 16S rRNA, or k. bases 0706-0732 of E. Coli 16S rRNA. The method as defined in claim 19, wherein said oligonucleotide probes correspond to the region including: a. bases 0163-0214 of E. Coli 16S rRNA, or b. bases 0176-0205 of E. Coli 16S rRNA, or c. bases 0163-0204 of E. Coli 16S rRNA, or d. bases 0437-0463 of E. Coli 16S rRNA, or e. bases 0641-0671 of E. Coli 16S rRNA, or f. bases 0821-0845 of E. Coli 16S rRNA, or g. bases 0195-0215 of E. Coli 16S rRNA, or h. bases 0156-0185 of E. Coli 16S rRNA, or i. bases 0829-0854 of E. Coli 16S rRNA, or j. bases 1107-1140 of E. Coli 16S rRNA, or k. bases 0706-0732 of E. Coli 16S rRNA.

36. The method as defined in claim 1, wherein said oligonucleotide probes correspond to the region including: a. bases 0163-0268 of E. Coli 16S rRNA, or W 61 b. bases 0391-0450 of E. Coli 16S rRNA, or c. bases 0631-0868 of E. Coli 16S rRNA, or d. bases 1102-1145 of E. Coli 16S rRNA.

37. The method as defined in claim 19, wherein said oligonucleotide probes correspond to the region including: a. bases 0163-0268 of E. Coli 16S rRNA, or b. bases 0391-0450 of E. Coli 16S rRNA, or c. bases 0631-0868 of E. Coli 16S rRNA, or d. bases 1102-1145 of E. Coli 16S rRNA.

38. The method as defined in claim 1 wherein each of said probes ia specific and complementary to mutually exclusive regions of Campylobacter 16S rRNA.

39. The method as defined in claim 19 wherein each of said probes is specific and complementary to mutually exclusive regions of Campylobacter 16S rRNA. The method as defined in claim 19 wherein each of said probes is specific and complementary to mutually exclusive regions of the Campylobacter rRNA sequence.

41. The method as defined in claim 1 wherein each of said probes is specific and complementary to mutually exclusive regions of the target nucleic acid sequence.

42. A method for activating a chemiluminescent molecule when usedin an assayfor Campyiobacter rRNA including: a. adding a first reagent to the test sample, said first reagent including an acid having a normality greater than 0.1N; and b. adding a second reagent to the test sample, aid second reagent including a base having a normality greater than 0.25N.

43. A method as defined in claim 42, wherein said acid includes HNO 3 and said base includes NaOH.

44. A method as defined in claim 43, wherein said acid is 1.ON HNO 3 and said base is 2.5 N NaOH. DATED: 29 April 1994 PHILLIPS ORMONDE FITZPATRICK Attorneys for: ,L; 9 S v 62