JPH044900A - Evaluation and analysis of hybridization for campylobacter rrna - Google Patents

Abstract

PURPOSE: To assay Campylobacter series by forming a specific hybrid complex containing a target nucleotide sequence and at least two labeled oligonucleotide probes and capturing the hybrid complex by a solid substrate.

CONSTITUTION: A hybrid complex containing a target nucleic acid sequence and at least two labeled oligonucleotide probes in which each probe is complementary and at least one probe is specific to a region of the target nucleic acid chain is formed by a solution hybridization. The hybrid complex is captured by a solid support and the target nucleic acid sequence is detected by label of the detected probe and the nucleic acid sequence is bound to the solid support by label of captured probe. Thereby, the target nucleic acid hybridized with both of detected probe and captured probe and the target nucleic acid bound to solid substrate is detected.

COPYRIGHT: (C)1992,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Applications) The present invention provides - oligonucleotide probes and immunochemical techniques for boat fishing, and the combination of such probes with immunochemical techniques for diagnostic and other application purposes. Regarding how to use it. More particularly, the invention consists of sandwich hybridization to detect the presence of one or more Campylobacter species and, in an extended assay format, to detect one or more target nucleic acids in a sample.

BACKGROUND OF THE INVENTION Campylobacter species are currently considered a major cause of acute bacterial enteritis. These spiral-shaped pathogens are known to thrive on the surfaces of the human intestinal tract, and although the disease is self-limiting, early treatment with antibiotics can shorten the duration of the disease and reduce the amount of stool Reduce excretion.

Since the discovery that Campylobacter is a cause of enteritis, biochemical methods have identified numerous strains of this bacterium, but the mechanisms by which these strains cause disease are unknown. Studies investigating the determinants of pathogenicity ascribed to Campylobacter include metastatic growth, attachment, invasion, and cytotoxin and endotoxin production. Labigne-Rousse
l) et al., 170(4)I), 1704-17
08° (1988).

The main pathogen of medically important enterocolitis is C. jejuni (
jcjuni), C,:Isu(coli), and C
, Laridis.

Campylobacter and other microorganisms present in the sample are
It is generally detected by culture technology.

Such techniques require that a suitably prepared viable sample be deposited on a suitable microbiological medium and subjected to environmental conditions favorable to promote the growth of the organism. The resulting colonies are then examined for morphological and biochemical characteristics.

In the early to mid-1970s, two new technologies were developed that, with further research and refinement, promise significant advances in medicine, particularly in diagnostic evaluation and analysis. One of these technologies is based on immunological mechanisms (the development and use of monoclonal antibodies) that depend on specific types of macromolecules and are limited by their properties. Other techniques are based on the manipulation of genetic material and include materials and methods for analyzing such portions. The genetic method is
Contains all the information contained in genetic material, DNA and RNA. DNA consists of two complementary strands of nucleotides. In its denatured form, the 6-strand has the ability to hybridize (anneal) with its complementary strand even in the presence of a large number of unrelated nucleotide sequences. Nucleic acid hybridization involves DNA/DNA hybridization in solution or on a solid support.

It can be carried out using various combinations of DNA/RNA and RNA/RNA. Older solid supports for hybridization include nitrocellulose and chemically treated paper. Southern, J., Mo1. Blo.

98 p., 503-517 (1975). Applications of such hybridization techniques involve the use of labeled nucleic acid molecules (probes), natural or synthetic, specific for abnormal nucleic acid molecules and etiological pathogens.

Techniques for synthesizing nucleic acid hybridization probes consisting of chains of deoxyribonucleotides or ribonucleotides are well known in the art. -For boat fishing,
To make a probe, target DNA is isolated from a cell, denatured to form a single strand, and copies of certain portions of that strand are isolated or synthesized in the laboratory and labeled.

Upon exposure to a complementary strand of target DNA or RNA in a sample, the labeled probe hybridizes to the complementary strand of target DNA or RNA.

Probes can be labeled using radioisotopes, fluorescent molecules, luminescent molecules, enzymes or immunochemical molecules. The label on the probe is then detected to detect the target DNA or RNA of interest.

Rashtchian European Patent Application No. 873DO5 [i9.8] describes oligonucleotide chains that hybridize Campylobacter IGS rRNA. A quantitative evaluation analysis using a solid support (plastic tube) with a label (avidin) is described, in which a labeled (biotin) DNA probe hybridized to Campylobacter 16S rRNA is transferred to the solid support (plastic tube). Unbound label on the support is detected by determining a+++ (the amount of biotin bound to the tube is estimated from the absorbance after treatment of the tube with avidin).

Goodson European Patent No. MM 87302354
．． No. 3 uses at least two labeled oligonucleotides, a colorimetric liquid hybridization assay for detecting nucleic acid chains, and a solid support (microtitration vessel) for subsequent separation. describes the capture of hybrid complexes. Restrictive site characteristics and N. gonorrhea of grouped cell anemia
describes a method for detecting hair surface antigens for e).

Hogan et al. European Patent Application No. 87310363.
No. 4 describes a method for preparing oligonucleotide probes complementary to variable regions of rRNA, selected to exhibit specificity for non-viral organisms. Probes that exhibit specificity for Campylobacter 16s rRNA are disclosed.

US Pat. No. 4,785,080 to Rashtehian et al. describes a DNA probe (900-1500 nucleotides) that can hybridize the DNA of at least 80% of Campylobacter jejuni bacteria. Denatured bacterial DNA is immobilized on a binding support, and the bacterial DNA is then hybridized with a labeled probe. Another method using unlabeled probes has been described, in which the contacting and detection steps are performed by sandwich hybridization.

US Pat. No. 4,745,181 to Law et al. describes the use of chemiluminescent labels in specific binding assays, such as immunoassays or nucleic acid hybridization assays. Polysubstituted arylacridinium esters are described.

US Pat. No. 4,672,040 to Josephson describes the use of magnetically responsive particles in nucleic acid hybridization evaluation assays. magnetic particles bound to nucleic acids are dispersed in a reaction mixture containing the molecule to be liberated;
After hybridization, particles with bound molecules are separated from the reaction mixture. The hybridized molecules are then separated from their magnetic particles.

International application PCT/CB86100170 of Hill et al. discloses that for separation purposes - heavy strand DNA or l? The use of magnetic or magnetizable materials coated with substances that can be sealed to the NA is described. After separation, the substance bound to the magnetic or magnetizable material is contacted with a probe and the presence of the single strand substance is detected by hybridization.

Rashtchian et al., Cl1n,
Chem, 33/9p, 1526-1530 (198
7) Immunological capture method for nucleic acid hybrids;
and its application to non-radioactively labeled DNA probe evaluation analysis. A synthetic NA probe complementary to Campylobacter 16S rRNA was labeled with biotin and was used to synthesize RNA probes obtained from bacterial cell lysates or Bommer RNA.
to hybridize. After hybridization,
The hybrid is captured with an immobilized anti-DNA:RNA antibody and detected with a biotin-based probe conjugated to streptavidin-horseradish peroxidase. This evaluation assay detected 70,000 Campylobacter from pure culture samples.

European Patent Application No. 82303701.5 of Heller et al.
No. describes a photoemission polynucleotide hybridization evaluation assay. A solid support method is described in which a target mm-strand polynucleotide is immobilized on a suitable support and the immobilized sample is treated with a
Label (peroxidase or iron porphyrin derivative)
the immobilized hybrid consists of contacting the labeled-heavy strand polynucleotide moiety, separating the unhybridized mm strand polynucleotide moiety, exposing the immobilized hybrid to a means for exciting a photolabel, and detecting its photoresponse.

Hansen European Patent Application No. 84306513.7
No. 2006, the authors form a biotinylated nucleic acid probe bound to an avidin-coated solid support, and convert the solid support bound probe into a hybridization product of an enzyme-labeled nucleic acid and a target nucleic acid. A sandwich hybridization evaluation analysis is described.

US Pat. No. 4,563,419 to Ranki et al. describes a competition-step sandwich hybridization evaluation assay for detecting target microbial nucleic acids. A first nucleic acid probe immobilized on a solid support is hybridized with a target nucleic acid sample and a labeled second nucleic acid probe. Following hybridization, the label associated with the hybrid complex bound to the solid support is detected.

Malcolm's international application POT/GB85100591 describes the use of solid supports (polymer beads) as carriers for immobilized nucleic acid moieties and unimmobilized second labeled nucleic acids.
describes a sandwich hybridization reaction (two-night culture) using

British Patent No. 2 of Sodcrlund,
No. 169.403 discloses a solution hybridization method for identifying nucleic acids. The detection (radiolabeled) and capture nucleic acid probes are hybridized with the target nucleic acid sequence prior to capture to a solid support (affinity chromatography).

Snitman International Application PCT/LIS8610128
No. 0 describes solution hybridization, which involves immobilizing the resulting hybrid complex on a solid support, followed by a second hybridization of the target nucleic acid chains. An improved method uses a distinct second probe during the first hybridization step to increase capture to the solid support.

Cohn, US Pat. No. 4,851,330, describes a method for detecting, identifying, and quantifying groups of non-viral organisms by hybridization evaluation analysis.

Other patents that may be important are listed below.

Chisvel 1 U.S. Patent Nos. 4 and 7
No. 16,106, [Detection of Polynucleotide Chains], U.S. Patent No. 4 to Dal tagupta
, No. 670゜380, [Evaluation Analysis Using Labeled Nucleic Acid Probes], International Application PCT of Gingeras et al.
/US8710L9G6, “Nucleic Acid Probe Evaluation and Analysis Method and Composition”, Heller European Patent Application No. 11B1111191.
No. 5, “Method for Increasing the Sensitivity of Nucleic Acid Hybridization Evaluation Assays,” Kourilski et al., U.S. Pat. No. 4,581,333;
“Method for Detecting Nucleic Acid or Reactants for Applying the Method”, Miller European Patent Application No. 85309224.5, “Polynucleotide Hybridization Evaluation Analysis Using Catalytic Luminescence”, Nogueira et al., US Patent No. 4.80
No. 1,530, “Nucleotide Hybridization Evaluation Assay for Protozoan Parasites”, European Patent Application No. 85105130.0 to Laboni et al.
No. 4,797,355 to Stavinski, “Hybridization Method for Detecting Genetic Material,”
``Method for Attaching Polynucleotides to a Support'', Taper et al., U.S. Pat. No. 4,61119,295, ``
Test for Salmonella”.

This technique utilizes the rapid kinetics of liquid hybridization to detect one or more bacterial genera or species, where hybridized products can be separated from unhybridized probe and sample mixtures. There remains a need for timely, simple and sensitive methods.

DEFINITIONS As used in this specification and claims, the following terms:
Define as follows.

1. Bacteria A member of the phylogenetic group Eubacteria, which is considered to be one of the animal, plant, and mineral kingdoms.

2. Complementarity Hybrid double-stranded DNA:DNA, RNA:RNA or DNA due to hydrogen bonding between Watson-Crick base pairs on each strand
: The property conferred by the base chains of mm strands of DNA or RNA that can form RNA. Adenine (^
) is usually complementary to thymine (T) or uracil (U), whereas guanine (G) is usually complementary to cytosine (C).

3. Hybrid Hybridization of two m by Watson-Crick base pairing or non-standard base pairing between complementary bases.
A complex formed between m-strand nucleic acid chains.

4. Hybridization A process, environment, and conditions in which at least two complementary nucleic acid strands combine (anneal) to form a double-stranded molecule (hybrid).

5. Kit A combination package of containers holding the evaluation analysis components necessary to perform a sandwich hybridization method for detecting the presence of one or more target nucleic acid chains in a sample. equipment necessary to conduct evaluation analysis;
Instrumentation and standard reagents may or may not be included as components of the kit.

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

7. Mutually Exclusive Regions Under the hybridization conditions of the preferred embodiment, two probes have the same nucleotide base chain on the target to the extent that hybridization of one probe prevents hybridization of the other probe. It means not competing for the same.

8. Nucleic acid probe Binds (anneals) with a complementary mm strand target nucleic acid chain to form a double strand molecule (
A single-stranded nucleic acid chain that forms a hybrid.

9. Nucleotide The lower unit of a nucleic acid, consisting of a 5° phosphate base, a 5-carbon sugar, and a nitrogenous base. In RNA,
5 carbon etc. is ribose. In DNA, the 5-carbon sugar is 2-
It is deoxyribose.

10. Oligonucleotide - A nucleotide polymer in which about 10 to 50 nucleotides are linked together.

11. Probe specificity A characteristic of a probe that indicates its ability to distinguish between target and non-target nucleic acid sequences. Probe specificity can be absolute (i.e., the probe can distinguish between target and non-target organisms) or functional (i.e., the probe can distinguish between the target organism and other organisms normally present in the sample). (can be distinguished) in some cases. A number of probe chains can be used in a wide or narrow range depending on the evaluation analysis conditions.

12. Sandwich immunoassay analysis An antibody for a first antigenic determinant is bound to a solid support, and the antibody bound to the solid support is transferred to a sample containing a substance having the first and second antigenic determinants. expose to

This removes antigenic material from the sample through the formation of a primary antibody-antigen complex bound to the support. This complex is then exposed to a second labeled antibody directed against a second antigenic determinant on the antigenic material to form an antigen-antibody sandwich that can be separated and detected. . The sandwich evaluation assay can also be modified to use other pairs of complementary molecules.

13. Target nucleic acid refers to the portion of a single-stranded polynucleotide having a nucleotide base chain corresponding to the genetic element whose presence in the sample is to be detected.

14. Sample: A purified sample containing one or more target nucleic acids.
or means a sample in unpurified form. The sample may be any DNA obtained from any physiological or laboratory source, including cells, biological tissue extracts, viruses, etc.
It can also be obtained from A or RNA (synthetic or natural).

SUMMARY OF THE INVENTION The main purpose of the present invention is to detect and quantify one or more Campylobacter species present in a sample, which is more timely and more specific than culture techniques. The objective is to provide an evaluation analysis method with higher sensitivity.

Another object of the invention is to provide a test kit for detecting and quantifying one or more bacterial genera or species present in a sample.

Another object of the present invention is that Campylobacter 1BS rRNA
The object of the present invention is to provide a plurality of oligonucleotide probes that are complementary and specific to.

Another object of the invention is to provide an oligonucleotide probe evaluation assay that does not require the use of radioactive materials.

Yet another object of the invention is to
An objective of the present invention is to provide an evaluation assay capable of quantifying Campylobacter in a sample containing bacteria.

Another object of the invention is to improve the detection of chemiluminescently labeled moieties, particularly chemiluminescently labeled oligonucleotide probes.

Still another object of the present invention is to provide a more timely, more specific, and more sensitive evaluation analysis method for detecting one or more nucleic acid chains present in a sample. It is.

SUMMARY OF THE INVENTION Under the hood, the present invention consists of a sandwich hybridization evaluation assay for Campylobacter species. This evaluation assay is a two-step method, the first step comprising a target nucleic acid chain and at least two labeled oligonucleotide probes, each probe being complementary, and at least one probe containing a target nucleic acid chain and at least two labeled oligonucleotide probes. Solution hybridization is involved to form hybrid complexes that are region specific. In the second step, the hybrid complex is captured by a solid support. The label of at least one probe (ie, the detection probe) is used to detect the target nucleic acid sequence, and the label of at least one probe (ie, the capture probe) is used to bind to the solid support. Only target nucleic acids hybridized with both the detection and capture probes and bound to the solid support can be detected in the assay format described above.

The evaluation assay format can be expanded to incorporate multiple units of oligonucleotide probes, each unit containing at least one capture probe and at least one detection probe. The capture and detection probes of each unit are complementary and at least one is specific for a region of target nucleic acid chains from the sample. Detection and quantification of one or more target nucleic acid chains, eg, one or more bacterial genera or species, in a sample is carried out similarly to the Campylobacter evaluation analysis described above.

A preferred form of evaluation assay uses a chemiluminescent molecule as a label for the detection probe, and the chemiluminescent molecule is an acridinium ester. This chemiluminescent molecule reacts with a suitable reaction reagent to generate a light signal, which allows the assay to achieve a sensitivity level for detecting the target nucleic acid sequence from a sample containing approximately 1xlO' bacterial cells per ml of sample. can be given.

The preferred evaluation assay of the present invention further includes a solid support, a label, and a magic 1 light evaluation analysis system (Ciba
Corning Diagnostics Corporation's equipment is used, but the Sepharo
se) Other solid supports, capture or detection labels, and devices can also be used, including 6B-CL. Such modifications may also improve detection sensitivity in accordance with the principles of the present invention.

DESCRIPTION OF PREFERRED EMBODIMENTS Oligonucleotide probes can be prepared synthetically, semi-synthetically, by recombinant DNA techniques, or from nucleic acids released from purified target nucleic acid chain samples. Also, Promega Corporation, Madison, Wl.
It is available from several sources including the United States.

The present invention includes methods for synthetically preparing DNA oligonucleotide probes for use in Campylobacter rRNA hybridization assays. Applied Biosystem Model 380B DNA Synthesizer User's Manual, Version 1.0, 198
July 5th. Applied Biosystem Model 381A DNA
Synthesizer User's Manual, Version 1.11, November 1985. Beaucage et al., Tetrahedron 1. ells, 22:1859-1882 (1
981), MaLLeucci and Carut
hers, J, Am, Chem, Soc, 10
3:3185-3191 (1981), Shinka et al., Tetrahedron Letts, 24+5843-5846
(1983). These methods can be used to prepare oligonucleotide probes for other target nucleic acid sequences, including various genera or pillars of bacteria to be tested in expanded forms of evaluation analysis.

Table 1 shows 13 oligonucleotide probes that are specific and complementary to Campylobacter 16S rRNA. The first column is the number assigned (as assigned by Promega Corporation) for each probe, and the second column is the number assigned to each probe (as assigned by Promega Corporation), and the second column is the number assigned to each probe (as assigned by Promega Corporation), and the second column is
) indicates the position of the probe corresponding to 16S rRNA,
The third column shows the base length of the probe and the fourth column shows the order of the probe.

Probe PM78. PH122 and PH138 are E
, it can be seen that they correspond to the same region of coli 16S rRNA.

The probes in Table 1 were tested for species specificity by hybridization methods. Campylobacter (n-133
) and non-Campylobacter organisms (n-73), the slot plot hybridization method was used to evaluate and analyze the specificity of the probes for various purified RNA samples released from non-Campylobacter organisms (n-73).

Kafatos et al., Nuc l. Ac4d Res, 7(6
) p, 1541-1552 (1970) and Torstay et al., Cold Spring Harbor Laboratory (1?, C,5chjIllke, e(1,)+)
, 231-238 (1982). RNA samples were released by the method shown in Table 2.

Table 3 shows detailed hybridization results for each probe and each strain of microorganism analyzed. Zero (0) indicates no hybridization;
(2) shows strong l\ibridization, and (1
) indicates weak hybridization and a blank indicates that no hybridization data were obtained for that particular strain. See symbols in Table 3. The data contained in Table 3 is summarized in Table 4. Based on the analysis of the results in Table 3, at least two such probes can be selected and used in the Campylobacter evaluation assay of the preferred embodiment. For example, probe PM78 may be selected for one type of evaluation analysis due to its ability to detect C. fetus. Probe P
M122 and PH138 are the same 16S as PH10.
Since they may compete for the rRNA region, they should not be selected for the same evaluation analysis format.

Of course, many of the same strains of microorganisms analyzed for probe specificity in Table 3 contain targets for oligonucleotide probe units to be incorporated into extended evaluation assay formats as described below. Similar, species-specific testing will be required before incorporating such oligonucleotide probes into expanded formats.

After testing oligonucleotide probes for species specificity, the ends of individual probes are modified with chemical groups that can form stable complexes with similar chemical groups. Nucl
, Ac1d Res, 13(7)p, 2399-241
1 (198B) and Ilaralambidls et al.
Nucl, Aeld Rcs.

15(12)I), 4857-4864 (1987). The chemical groups are selected so as not to interfere with the hybridization step of the assay. In the described examples and preferred embodiments, at least one probe is labeled with a binding partner to one or more first supports by techniques known in the art, including covalent bonding, and the at least one probe is labeled with one or more detection molecules.

The function of the support-bound partner is to facilitate the capture of the hybrid complex onto the solid support. The purpose and function of the solid support is explained below. The hybrid complex consists of a target nucleic acid chain annealed with at least two different oligonucleotide probes, at least one of which has bound thereto.
It has a counterpart that binds to one or more first supports. the solid support then has specific affinity for the partner immobilized thereon that binds to the first support;
including a partner that binds to one or more second supports.

The high specificity of biotin for avidin or strebavidin makes it very suitable for use as a support-binding partner in the present invention.

US Pat. No. 4,582,810 describes the formation of an avidin-biotin bridge in diagnostic compositions by association with solid support particles. Murasugi et al., DN
A3(3) p. 2Ei9-277 (1984)
An oligonucleotide labeled with biotin is described as a hybridization probe.

Immunoassay generally refers to a method of determining the presence or amount of a substance in a sample using antibodies specific for that substance. This evaluation analysis reaction is based on the antigenic substance (
The reaction requires the formation of an immunochemical complex between a hapten) and its respective antibody, and this reaction occurs simply by incubating the antibody with a sample containing the antigen.

Any of the reactants of the immunochemical complex may be immobilized on a solid support as provided in one embodiment of the invention. When a hapten is used as a partner for binding to a first support, such as dinitrophenol (DNP) in Example 1 below, antibodies against DNP (preferred monoclonal), and in particularly preferred embodiments anti-DNP ( 5111) serves as a partner for binding to a complementary second support.

A number of non-radioactive and radioactive labeling techniques and detection methods are known for detecting hybridized complexes.

The detector molecule is generally used for base pairing (Watson-Crick) between the oligonucleotide probe and the target nucleic acid chain.
Choose a method that will cause little interference. Examples of such molecules are radioactive, luminescent or fluorescent substances, enzymes producing luminescent, fluorescent or colorimetric substances, and others known in the art.

In a preferred embodiment, the detection molecule is a chemiluminescent molecule, more particularly an acridinium ester or a polysubstituted acridinium ester. The preparation and chemistry of polysubstituted arylacridinium esters are described in US Pat. No. 4,745,181, which is incorporated by reference. The light emission (signal) generated by the activation of chemiluminescent molecules can be detected using commercially available equipment, such as the MLA instrument (Ciba).
Corning Diagnostic Corporation)
It can be detected by

To increase detection sensitivity, one or more detector molecules can be attached to the oligonucleotide probe.

It is necessary that the first binding partner not bind to the probe labeled with the detector molecule. At least one oligonucleotide probe labeled with one or more detector molecules comprises one component of the oligonucleotide probe unit in the extended evaluation assay format of the invention. When multiple oligonucleotide probe units are incorporated into a desired evaluation assay format, more than one type of detector molecule per probe may be required to discern the signal or response generated by activation of the detector molecule. If more than one detector molecule is used, they can be detected sequentially by methods such as those shown in Example 1 below, or by using chemiluminescent labels with display signals at different wavelengths. . Alternatively, multiple detector molecules can be detected simultaneously.

The labeled oligonucleotide probes can now be used in hybridization evaluation assays for target nucleic acid chains.

Sample preparation requires lysis of the organism in question and protection of the released RNA from nucleases present in the sample. Lysis methods that lyse the desired intestinal pathogens without the need to lyse all cells in the sample are optimal.

Campylobacter cells are treated with 0.05% to 0.5% SDS.
Trls, EDTA buffer (pH 8-9)
) was found to dissolve at room temperature. Cells are SD
In the absence of S, Trls can survive in EDTA buffer.

Shigella (Shigel Ia) and Salmonella (Sa
lmoncl Ia) cells are more resistant than Campylobacter. To quantitatively lyse these organisms, SDS
It was necessary to either increase the a degree (0.25%) or increase the temperature.

Tris, 0.0% in EDTA buffer (pu 8-9).
Lysis with 25% SDS for 10 minutes at room temperature was the preferred method for cultured Shigella, Salmonella and Campylobacter cells. However, this method does not protect RNA released in stool samples.

Other steps are required to recover the RNA released from the stool sample. A preferred method combines SDS lysis with a heat inactivation step (75°C for 10 minutes) followed by filtration (LID/x (Genex) 25 μm polyethylene).
It is to do.

Other nuclease inhibitors were tested, but none were necessary other than SDS and heat inactivation. For example, 5-10mM VR
Contains C (vandyl ribonucleoside complex), 6
RNA is stabilized even if the bacterium is lysed at 5°C or at 75°C without VRC.

In the present invention, each of the oligonucleotides is specific and complementary, at least one of which is specific for a target nucleic acid chain suspected to be present in the sample.

Synthetic oligonucleotide probes include, for example, 5S,
It can be complementary to and specific for any variable region of rRNA, including 16S or 23S rRNA. In the above method for detecting and quantifying the presence of Campylobacter species in a sample, as listed in Table 1,
specific for and complementary to mutually exclusive regions of Campylobacter 168 rRNA.

Hybridization conditions necessary to achieve annealing of the target nucleic acid strands, if present, and at least two different oligonucleotide probes, many of which can be adjusted to increase the efficiency of the assay. Determined by numerous variables. These variables include the nature of the label attached to the probe, the linkage of the probe, the size of the probe (in base pairs), and the release/release of the target nucleic acid linkage.
Methods of preparation, and duration and temperature of hybridization are included. It is also true that technical experience influences the efficiency of test evaluation analysis. - Provide test kits that use a set of evaluation analysis components and use such kit components in combination with automated equipment (MLA system, Ciba Corning Diagnostic Corporation) to assess these variables. It is also one of the features of the present invention to reduce or make it more regular.

Fixation and separation of the hybrid complex from the sample mixture and excess unhybridized probe are
It is carried out using a solid support. One of the advantages of a solid support is that multiple binding partners can be immobilized thereon. Solid supports used in hybridization evaluation assays include plastic tubing, microtiter vessels,
There are known materials that are inert to cross-linked dextran, porous silicate glasses, magnetic particles coated with cellulose derivatives, nitrocellulose filters and evaluation analysis components.

In a preferred embodiment, paramagnetic particles (PNPs) are used as solid supports. This type of paramagnetic particle is described in US Pat. No. 4,554,088.

The partner that is bound to the second support can be immobilized on the PMP by a number of techniques, depending on its nature. If the partner to be bound to the second support is an antibody, the antibody can be bound to the PNPs by using glutaraldehyde as a binding agent. Reichlin, Method “Enzy,” 70p, 159-
1 [15 (1980).

In a preferred embodiment, the second support binding partner functions to capture the hybrid complex to the solid support. In pairs, the complex is separated from the sample mixture and excess unhybridized probe using a magnetic field. (MLA Rack, Ciba Corning Diagnostics Corporation) Although other methods for separating sandwich hybrid complexes have been introduced in the art, the MLA Rack has the advantage of being able to process multiple samples simultaneously.

EXAMPLES Example 1 A volume of Campylobacter jejuni cells (1×10 to 4×10
6), 50 gM Trls (Sigma Chemical Co.),
pH9,0,0,6M NaCI (Sigma Chemical Co.),
60a+M Sodium Citrate (Mallinkrodt)
, pl+ 7.5 and 100 ul of a 1:50 dilution of feces containing 0.05% 5DS (Sigma Chemical). Cells for sample preparation were lysed by heating to 95°C for 5 minutes. The released rRNA was 0.9 pmol (pI
lol) labeled oligonucleotide probe (
DNP-PM7 g, D labeled at its 5° end
NP) and chemiluminescent substance (acridinium ester (
0.2 pmol of a labeled oligonucleotide probe (PM23B) labeled at its 5° end with 8E) and at its 3° end with 32P at 58°C.
Time/1 hybridization was performed.

The nucleotide chain of PM78 is 5°-TCT GCC
TCTCCCTCA CTCTAG ACT ATG
Consists of AGT T-3°.

The nucleotide chain of PM238 is 5°-GCCTTC
GCAATG GGT ATT CTT GGT GA
It consists of T-3°.

Following hybridization, a 10 ul aliquot was added to 50 ug of paramagnetic particles (PNP) to which anti-DNP antibody (mouse monoclonal) was covalently attached. After culturing at room temperature (23°C) for 0.50 h, PNP
Bound Santoy-Sochi hybrids were separated from excess unhybridized probe and sample by separation in a magic dry rack (Ciba Corning Diagnostics Corporation), and the supernatant was removed. PMP 0.8M
NaCl, [io mM sodium citrate, 10
aM Tris, pH 7,0,50d EDT^.

(Sigma Chemical Company), 0.1% bovine serum albumin (BSA) (fils, fraction ■) and 0.02T
Ween-2 (Sigma Chemical Co.) was washed twice and then redispersed in 100 ul of distilled water. The solid support also captures unhybridized probe labeled with the capture molecule; It can be seen that the unhybridized probe labeled with the detector molecule is not captured.The bound sandwich hybrid is measured in relative light units (RLU
), the denaturing reaction reagents (Reagent 1 contains 1.0 N in a 0.5% solution of H2O2) INO3 and Reagent 2 contains 0.5% of a surfactant (Arquad)
(2.5 N HNO3 in solution) (see also Example 6) (Ciba Corning Diagnostics Corporation), as well as liquid scintillation counting of the sample (Atom Light Liquid Scintillation Cotscutil, New England Nuclear). Detected by. Campylobacter rRNA is found in IQ in samples.
, detected above the background with a cell coefficient on the order of OOO Campylobacter cells. FIG. 1 shows the relationship between the two labels and the concentration of Campylobacter cells in the sample. For comparison,
Purified E. coli rRNA (Pharmacia) was similarly hybridized, captured, and detected (without adding feces), but gave no light signal above the background signal.

The background signal was a chemiluminescent reaction (denoted RLυ) observed from a sample treated similarly but in which the bond to be labeled with PNP was closed with beta-analine DNP. Conversion of signal to cell count was calculated using the following formula.

The concentration of Campylobacter jejuni cells in the dispersion can be determined by quantitative culture or by using bibenzimidazole dye (Hoechst 33).
258) by fluorescence evaluation analysis. The cell dispersion was diluted 106 and 108 times in ice-cold sterile water. 5 samples of diluted solution (10 and 100 ul)
The cells were inoculated onto Pulsella agar containing 10% horse blood or Trypsoia agar containing 10% sheep blood. The board was placed in a microaerobic environment (5% 0□, TOXCO 8H
N2) Cultured at 37°C in medium temperature.

Colonies were counted after 48 hours of culture. Fluorescence measurement of DNA concentration of Cambilobata jejuni was performed using Bechist 33.
258 dye was used. The DNA concentration in this dispersion was determined by storing the cell dispersion and dye in the dark at room temperature (23°C).
It was measured by culturing for 0.50 hours. Fluorescence readings were compared to a lambda DNA standard curve. Calculation of the number of cells/ul was done based on the genome size of C. jejuni.

Example 2 Volume of Campylobacter rRNA (100 attomoles (aI)
Ilol) - 100 fmol (fmol) to 500
ul of 0,6M sodium chloride, 60mM sodium citrate, 10mM Tris, pH 8,0,
The 5° end was incubated with a chemiluminescent molecule (A
E) 0.25 piol of the oligonucleotide probe (AP-PH10) labeled with E) and 0.5 p of the oligonucleotide probe (AP-PH10) labeled at the 5° end with a chemiluminescent molecule (AE).
mol of oligonucleotide probe (AE-PM23
8) and 1.0 pool of oligonucleotide probes (5
Hybridization was performed with end-labeled biotin-PH10 (source of biotin was Aldrich Chemical). PH10(7) nucleotide linkage is 5'-G
TA CCG TCA GAA TTCTTCCCCT
AAG AAA-3°.

The hybrid complex has a partner (
Avidin) was immobilized on 50 ug of PNP.

The PNPs were separated and separated as described in Example 1.
Washed. For detection of captured hybridized complexes,
To obtain the highest sensitivity, chemiluminescent reaction reagents were modified as described in Examples 1 and 6. The result is RLU
Shown as Experiment/RLU background, where RLU background is the chemiluminescent reaction observed in the absence of added Campylobacter rRNA, indicating that approximately 100 amol of Campylobacter rRNA was detected. See Figure 2.

volume of Campylobacter rRNA (100 amol-10
0 fagof) at 65°C for 1.0 h, 500 ul
(same buffer as in Example 2) in which DNP acts as a partner for binding to the first support.
lol labeled oligonucleotide probe (DNP-P
M238), and 0.8 pa+ol of a labeled oligonucleotide probe (biotin-PH10), with biotin acting as the molecule binding to the second support. Following hybridization, the hybrids were first captured on PMP and then labeled with a labeled chemiluminescent molecule (avidin-AE) (advanced form) or first labeled with a chemiluminescent molecule (avidin-AE). AE) and then captured onto PMP (inverted form H). In another format, an oligonucleotide probe (biotin-PH10) labeled with a molecule that binds to a second support is combined with a chemiluminescent molecule (biotin-PH10) labeled with a chemiluminescent molecule (
Labeled in advance with avidin-AE). This adduct, a labeled chemiluminescent molecule and a labeled oligonucleotide probe (avidin-AE/biotin-PM7)
B> was hybridized with Campylobacter rRNA as well as a probe labeled with a molecule (DNP-PM238) that binds to the first support. In forward mode, 50
Solid support labeled with ug (5H1-PNP)
Hybrids were captured by culturing at room temperature (23°C) for 70.50 hours. Then, the PNPs are separated,
Wash as described in Example 1, about 5. Bx10
A chemiluminescent molecule labeled with 6RLII (avidin-A
E) in 100 ul of buffer.

After 2.0 hours, the PNPs were again separated, washed, and redispersed to activate the chemiluminescent molecules.

In the inverted format, a labeled chemiluminescent molecule (avidin-AE) of approximately 5.0x107 RL was added to each hybrid solution and incubated at 65°C for 0.25 hours. The labeled hybrids were then incubated with 50 ug of labeled PN.
P(5111-PNP)Te room temperature (23℃) 70.50
Captured by culturing for hours. Examples 1 and 2
Samples were treated in the same manner as above to activate chemiluminescent molecules. The results of these evaluation analyzes were expressed as signal/background relative to the total amount of rRNA, where the background is the signal observed in the absence of rRNA, as shown in FIG.

Example 4 A sandwich hybrid consisting of Campylobacter jejuni rRNA and two derivatized and labeled oligonucleotide probes, one for capture and one for detection, was mixed with 1 out of 60 mM sodium citrate; 1
0mM Tris, 0.6M Sodium Chloride, 50
Total amounts of Campylobacter rRNA present in mM EDTA and 0.05% SDS (hybridization buffer) and a doubly labeled probe for detection of 160fn+ol (5°-AE, 3'-"2P-
PH238) and for capture, 2. Molecule attached to the first support of Opfilol (5°-DNP-PH10)
oligonucleotide probe labeled with or 2.
The molecule that binds to the second support of OpIIlol (5゛-
It was prepared by combining a probe labeled with biotin-PM7g) and then culturing at 56°C for 2.0 hours. Following hybridization, 10u
1 aliquot of 90ul of 60H sodium citrate, 16ff of 1M sodium phosphate, 0.72M
Sodium chloride, 0.08% BSA, 0.02%
Tveen-20, and 50 ug of capture probe specificity solid support (added to PM circles in 0.04% sodium azide, pH 7.2 (phosphate evaluation assay buffer). Capture specificity is a labeled oligonucleotide probe (DNP-PH10 or biotin-PM7).
g) to capture sandwich hybrids containing respectively labeled PNP, 5 former anti-DNP-
P) determined by binding molecules covalently attached to IP or avidin-pip. Non-specific binding of the detector molecule to the solid support (PNP) can be avoided by adding excess beta-analine-DNP or biotin to the PNP, respectively, to close all binding sites before adding the sandwich hybrid. Assessed by reaction.

After 0.50 hours of incubation at room temperature (23°C), bound sandwich hybrids were separated from excess unhybridized probe and sample as described in Example 1.

PNPs were washed twice with phosphate buffer and redispersed in 100 ul of distilled water. Bound sandwich hybrids were detected by chemiluminescence reaction using standard MagicDrite reagent and liquid scintillation counting. The results of these evaluation analyzes are shown in Table 5.

Example 5 Sandwich hybrid described in Example 4 (PNP
), 0. I M NaCl, 10
．． OtnM Tris, 1. OmM EDTA, p
It was analyzed by chromatography on a Sepharose CL-6B (Pharmacia) column (Pasteur pipette) paralleled with a buffer pH 7.5 containing H7.5. Oligonucleotide probes hybridized to Campylobacter rRN^ eluted into space, and non-norhybridized oligonucleotide probes were retained in the column. This column fraction was analyzed first by chemiluminescence reaction and then by liquid scintillation counting. The results of these evaluation analyzes are shown in Table 6.

These data indicate that the extent of hybridization is similar whether assessed by chemiluminescence or liquid scintillation counting. PMP
In contrast to Example 4, where the sensitivity for detection of the sandwich hybrid captured above is significantly better by liquid scintillation counting than by chemiluminescence reaction when analyzed by chromatography, the sensitivity of these two detection methods is The chemiluminescent reaction was found to be equivalent or slightly better.

Example 6 Campylobacter rRNA of 0.5p[l1o1, 2.
Opmol labeled oligonucleotide probe (biotin-PH10) and 1.0 pmol labeled oligonucleotide probe (5'-AE-P
Sandwich hybrids were formed with Campylobacter rRNA by combining H238) in 100 ul of hybridization buffer and incubating for 2.0 hours at 56°C.

Resulting hybrid complex and labeled PMP
(avidin-PMP), 5. Mix in a ratio of Ofmol hybrid complex to 250 ug labeled PMP;
After culturing at room temperature (23°C) for 0.50 hours, hybrids were captured on a solid support. Bound sandwich hybrids were separated from excess unhybridized probe and sample as described in Example 1. P) IPs were washed twice with phosphate assessment assay buffer and redispersed in 100 ul of distilled water. Chemiluminescent molecules (AE), labeled oligomeric DNA probes (AE-P
H238) and sandwich hybrids were dispersed in distilled water and divided into 100 ul aliquots.

The following parameters related to the chemiluminescent reaction were tested as described in Examples 4 and 5 to increase the detection efficiency of the hybridization evaluation assay. The time dependence of the chemiluminescent reaction for the above solution was measured using standard MLA.
The conditions were measured by monitoring the output of the photomultiplier tube of the MLA instrument. It can be seen that in each sample, the activity reaches a maximum value within 1 second of the onset of the chemiluminescent reaction and then declines to background level readings within 2 seconds, indicating that all of the available light signal is recorded by the MLA during the standard integration. It was.

The first step in a chemiluminescent reaction is the formation of a chemiluminescent molecule (AE
) involves the attack of hydroperoxide ions on
Forms an electronically excited molecule, which reacts with reagent 1
MLA
The standard value of delay between injections of reaction reagents in sequence is 0.1
It was measured by changing the time from seconds to 10 minutes. The results of these evaluation analyzes are summarized in Table 7, and it was found that the signal increased only to a negligible extent with respect to changes in unit time.

Matrices of reaction reagents with component concentrations equal to or 10 times greater than standard reagents were tested for their ability to elicit chemiluminescent reactions of AC-labeled oligonucleotide probe solutions. All denaturing reagents tested increased chemiluminescent activity, but the acid and base normality was increased by a factor of 10 (denaturing reagents) relative to the standard reagent, and the hydrogen peroxide and surfactant concentrations were When unchanged, the signal reached its maximum value. Under these conditions, the chemiluminescent signal detected from the sandwich hybrid in solution by a photomultiplier increases by a factor of 2 over that observed with standard reagents, with a 13-fold increase in the captured sandwich hybrid. observed against. The results of these evaluation analyzes shown in Table 8 demonstrate that the conditions that elicit chemiluminescent responses from AE-oligomer sandwich hybrids bound to paramagnetic particles are comparable to those observed for the same complexes in solution. It is shown that.

Example 7 An aliquot of the dual-labeled sandwich hybrid formed with biotin-PH10 in Example 4 was captured and a denaturing reagent as described in Example 6 was used in the MLA to detect the chemiluminescent reaction. Processed as previously described. Following the chemiluminescence reaction, samples were detected by liquid scintillation as described above. The results of these evaluation analyzes are summarized in Table 9. The detection sensitivity of the chemiluminescent reaction using the denaturing reagent increased significantly compared to the standard reagent, while the detection using liquid scintillation counting did not change much.

Example 8 1.0 pmol Campylobacter rRNA, 1.0
pmol labeled oligonucleotide probe (biotin-PH10) and 2.0 pmol labeled oligonucleotide probe (DNP-PM2).
Sandwich hybrids were formed with Campylobacter rRNA by combining 3g) in 100 ul of hybridization buffer and incubating at 65°C for 1.0 hour.

60 mM Sodium Citrate, 10.0 IIIM Tris, 1.0 mM with 0.05% SDS
EDTA, 0.6 M sodium chloride, 0.1%
BSA, 0.01% sodium azide and 0.02
Dilutions of this sandwich hybrid were prepared in % Tween-20, pH 7,4 (Trjs evaluation analysis buffer) to a final concentration of 2.2xlO to 2.2xlO"' M. A sample of the diluted hybrid (0.45 ml ),
10-” to 10-16 moles of labeled PMP (5111-PNP) in 501 Tr1s evaluation assay buffer.
combined with. To eliminate non-specific binding to PNP, add excess beta-alanine-D'N to close all binding sites before adding the sandwich hybrid.
The reaction was evaluated in parallel with the addition of P to PMP. Room temperature (2
After culturing at 3°C for 0.50 h, as above,
Bound sandwich hybrids were separated from excess unhybridized oligonucleotide probe and sample. PMP in Tris evaluation assay buffer at 1
6.6x106 RLL and then the PMP
The chemiluminescent molecules of I were redispersed in 100 ul of Tris assay buffer containing a covalently linked second binding partner (avidin-AE) and incubated for 2.0 hours at room temperature (23°C). Fixed sandwich hybrids were labeled. The PMPs were washed twice in Tris evaluation assay buffer to remove unbound avidin-AE and then redispersed in 100 ul of distilled water.

The folded samples were processed with standard and denaturing reagents in the MLA instrument. The results of these evaluation analyzes are shown in Table 10.

By combining the results of Examples 4 and 5, Sandinch bound to a solid support containing a chemiluminescently labeled oligonucleotide probe was more effective in detection than the same Ibrid in solution using standard reaction reagents. is low, ie, only a portion of the total hybridized chemiluminescent labeled oligonucleotide captured on the solid support is activated by the standard reaction reagent.

In Example 4, detection by liquid scintillation counting is arbitrarily defined as the hybrid input required to yield an S/B value of 2 for detection performed by chemiluminescence reaction using standard reaction reagents. Sensitivity is significantly improved. However, in Example 5, the sensitivity with the rain detection method is comparable when the same hybrid is analyzed following chromatographic elution that allows solution phase separation of hybridized and unhybridized probes.

In the first step of the chemiluminescent reaction sequence, a certain amount of reaction reagent 1 is injected for a short period of time (1-2 seconds), and the hydrogen peroxide contained in the solution reacts with the chemiluminescent molecules (AE), causing electronic Forms an excited molecule (N-methylacridone). Then, after a short time (0.1 s), the same amount of the second reagent is briefly injected (1.2 s), and once the pH becomes basic, the excited molecules undergo a reversible reaction involving the emission of light. cause a reaction. This emitted light signal is detected by a photomultiplier tube that is physically close to the sample, and the microprocessor of the MLA instrument adjusts the output of the photomultiplier tube for a certain period of time (starting from the addition of the second reagent). 2,0
seconds). The results of Example 6 showed that the chemiluminescence signal was 2
Within seconds it drops to the background, indicating that all available signals have been recorded. These include chemiluminescent reactions of chemiluminescent molecules alone, chemiluminescent molecules covalently bound to oligonucleotide probes, sandwich hybrids labeled with chemiluminescent oligonucleotide probes in solution, and chemiluminescent molecules bound to solid supports. This applies to luminescent oligonucleotide probe label sandwiches. Therefore, the low detection efficiency of solid phase-bound chemiluminescent oligonucleotide probe-labeled sandwich hybrids observed with standard reaction reagents was not due to delayed activation of chemiluminescent molecules (AEs) in the complex. .

During the MLA step, the amount of excited molecules (N-methylacridone) generated from the chemiluminescent oligonucleotide probe-labeled sandwich hybrid attached to the solid support is increased in the first reaction reagent, thereby In an attempt to increase the signal generated when adding reaction reagents, the incubation time of the sample in the first reaction reagent was increased by a factor of 6000 (0, from 1 second to 10 minutes). However, only a small increase in the generated signal was observed and does not account for the order of magnitude difference in detection sensitivity due to chemiluminescence versus liquid scintillation counting.

Finally, the signal generated from a chemiluminescent oligonucleotide probe-labeled rRNA sandwich hybrid bound to a solid support was significantly higher when the normality of the reaction reagents was increased tenfold, ie, with denaturing reagents.

Of course, various other modifications will be apparent to, and may be practiced by, those skilled in the art without departing from the scope and materials of the invention. However, the scope of the claims is not limited to the above description, and the claims include all features that a person skilled in the art would treat as equivalent. It encompasses all characteristics. Moreover, the examples are provided here for illustrative purposes and are not intended to limit the invention.

Table 1 II LiUI'LiA'1 Table 2 RN^ Purification Method 1. Add 0.1 g wet weight of cells to bead beater tube.

2. Discard the supernatant. Re-disperse the cells in 700 ul of Buffer 1 using a sieve roll.

3.50ul of 20% SDS and 0.1-0.15
Add mm glass beads to about 1/4 of the way up the tube.

4. Add equilibrated phenol to fill tube 5.

5. Close the cap and beat with a bead beater for 4 minutes at room temperature.

6. Soak the tube in a water bath (60°C) for 15 minutes to remove proteins and degrade DNA.

7. Dry the outside of the tube and tap for another 2 minutes.

8. 300 ORPM (735xg
) for 5 minutes. Do not use the microfuge at high speeds. Alternatively, spin in a Speed Guac for 10 minutes without applying vacuum.

9. Remove almost all the aqueous phase (top phase) and interface to a new sterile 1.5 ml microfuge tube (i.e., leaving the beads and phenol behind).

10. Fill with phenol, then vortex for 1.5 minutes.

11. Microfuge LOOORPM (5220x
g) for 5 minutes. Remove the aqueous phase to a new tube and take a small amount of phenol if necessary.

12. Perform phenol extraction (steps IO and 11) two more times.

13. Perform two phenol/chloroform (phenol/chloroform/isoamyl alcohol 25:24:l) extractions as in steps 10 and 11, but always leaving behind the interface and bottom layer after the last extraction.

14. Add 0.1 times the amount of 38 sodium acetate, pH 5.2, to the remaining aqueous phase.

15. Divide into tubes (50 ul per back).

16. Add 2 volumes of cold ethanol. Swirl briefly. Precipitate in one apparatus at -70°C. Long-term storage of the preparation should take place in this form and at this temperature.

17. -L9) -14.00 ORP of sedimentation tube
Microcentrifuge at M (16,000xg) for 10 minutes. The ethanol supernatant is removed and the pellets are dried in a speed guac (usually 5 minutes in vacuo).

18. To determine the optical density, redisperse the dried pellet in 300 ul of DEPC treated water. This sample was diluted 1:10 in DEPC water and
and measure the absorbance at 280 rim. Discard the solution in the cuvette and store the remainder at -70°C. This is the working solution.

a. Grow bacterial fusion strains on plates to prepare cells. Each plate yields 0.1-0.2 g of cells depending on the strain.

1) "Shave" the plate with a sterile razor blade and transfer the cells to a preweighed 501 plastic conical centrifuge tube.

2) Add approximately 10 1 volume of LB medium and swirl to redisperse the cells.

3) In a Beckman TJ-6R table top centrifuge at 2400 RPM (1200x
g) for 10 minutes.

4) Remove the supernatant.

5) Measure tubes and cells to determine wet cell weight.

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

7) To isolate RNA, add 0.1 ml of wet cells (
i.e., 1 g) into a bead beater tube. Spin at 3.00 ORPM (735xg) for 5 minutes in a microfuge. Discard the supernatant. This sample is processed as described above.

8) Transfer the remainder to Tl+-4 in a tabletop centrifuge.
Rotate at 240 ORPM (+200xg) at 0-tar.

9) Exclude liquid. Store cells at 0°C.

Another method to obtain bacteria is: 1) Add 1-2n+l LI3 to each plate. ``Redisperse the cells in the LB by PET manipulation or by removing the cells with a bent glass rod.

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

3) Perform steps 3 to 9 above.

b. To equilibrate the phenol, add an equal volume of the desired buffer, mix vigorously for approximately 30 minutes, and in a table-top centrifuge at 50 ORPM (50
xg) for 5 minutes to separate the phases. For the twice equilibrated phenol, remove the aqueous phase from the parallelized phenol and repeat.

C. For phenol/chloroform/isoamyl alcohol, mix phenol with equalized sugar volume and chloroform/isoamyl alcohol premixed at 24:1.

TABLE 3 (5 of 5) The first column shows each strain number used by Promega Corporation. The second column indicates the genus and species name of each microorganism. The third column shows the evaluation of the state of rRNA purified from each strain after agarose gel electrophoresis. Two main bands are expected for each sample, the upper band being 23S
It is thought to be rRNA, and it is thought to be the lower 183 rRNA.

Depending on the guidelines below, other bands may be observed or no band may be observed.

- Both bands are present and at normal strength 2 - The upper band is not very visible 3 - The upper band is barely visible 4 - The upper hand is absent 5 - The lower band is not very visible 6 - The lower band is barely visible 7 - There is no lower band 8 - The specimen has one other band 9 - The specimen has two or more other bands Display rGel J If the column containing a large number has a large number, the above case applies.

In the rest of the table, (0) indicates no hybridization, (2) indicates strong hybridization, and (1) indicates weak hybridization.

Blank boxes indicate either no testing or evaluation of specific probes with specific bacterial strains.

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Drive

[Brief explanation of the drawing]

Figure 1 shows the results of the comparative label evaluation analysis for Campylobacter described in Example 1, and Figure 2 shows the results of Example 2.
FIG. 3 shows the results of the magicpyrite sandwich hybridization evaluation analysis for Campylobacter rTINA described in Example 3, and FIG. Show the results. G; Body V- Figure 2 Kang 0 Rono foot RNA 1: Te [Surumashi 1) Light meter A 1h Li rod + RLU ... stand... (p Kanko ° O 8 end 9vRNA ;) tl Rekanhi Roba'kuta! r, 1EIW begging

Claims (1)

[Scope of Claims] 1) A method for evaluating and analyzing Campylobacter rRNA, which includes: (a) consisting of cells of one or more cell types and estimated to contain Campylobacter cells or Campylobacter rRNA;
(b) releasing rRNA from cells of the sample; (c)
Campylobacter rRNA in the sample, if present.
is hybridized with at least two labeled oligonucleotide probes to form a hybrid complex, each of which is complementary and at least one of which is a Campylobacter 16S rRNA.
has a nucleotide linkage that is specific for the region of
labeling at least one of the probes with a partner that binds to one or more first supports; and labeling at least one of the probes with one or more detector molecules; d. capturing the hybrid complex on a solid support;
forming a sandwich composite, the solid support comprising:
having one or more second support-binding particles immobilized thereon that are complementary to the first support-binding partner; binds to a partner that binds to the second support; e. separating the sandwich complex from the sample and excess unhybridized probe; and f. binding to the sandwich complex. A method characterized by detecting the presence of Campylobacter by activating a detector molecule. 2) The method according to claim 1, wherein the partner that binds to the first support is a hapten, and the partner that binds to the second support is an antibody. 3) If the oligonucleotide probe is a, 5'-[there is a gene sequence] -3', or b, 5'-[there is a gene sequence] -3', or c, 5'-[there is a gene sequence] ] -3', or d, 5'-[There is a gene sequence] -3', or e, 5'-[There is a gene sequence] -3', or f, 5'-[There is a gene sequence]- 3', or g, 5'-[There is a gene sequence] -3', or h, 5'-[There is a gene sequence] -3', or i, 5'-[There is a gene sequence] -3' , or j, 5'-[there is a gene sequence] -3', or k, 5'-[there is a gene sequence] -3', or l, 5'-[there is a gene sequence] -3', or 2. A method according to claim 1, characterized in that the gene sequence comprises at least two different nucleotide chains of the group consisting of m, 5'-[there is a gene sequence]-3'. 4) The method according to claim 1, wherein the detector molecule is a chemiluminescent molecule. 5) The method of claim 4, wherein the chemiluminescent molecule is an acridinium ester. 6) The method of claim 4, wherein the chemiluminescent molecule is dimethylacridinium ester. 7) A method according to claim 1, characterized in that the solid support is a paramagnetic particle. 8) activating said detector molecules by adding a first reagent solution and then, following an incubation period, adding a second reagent solution to initiate a detectable photoreaction; A reagent solution of 1.0N in a 0.5% solution of H_2O_2
2. The method of claim 1, wherein the second reagent solution is 2.5N NaOH in a 0.5% surfactant solution. 9) The method of claim 8, wherein the surfactant is Arquad. 10) Further comprising the step of quantifying the number of Campylobacter cells in the sample, the sensitivity of the test evaluation analysis is
2. The method of claim 1, wherein approximately 1 x 10^4 bacterial cells can be determined per milliliter. 11) the partner binding to the first support is avidin/strepavidin or biotin, respectively;
and the partner binding to the second support is biotin or avidin/strepavidin. 12) The method according to claim 2, wherein the partner that binds to the first support is dinitrophenol, and the partner that binds to the second support is an anti-dinitrophenol antibody. 13) The method according to claim 1, wherein the detection molecule is an enzyme. 14) The method according to claim 1, wherein the partner that binds to the first support is an antibody or an antigen, and the partner that binds to the second support is an antigen or an antibody, respectively. . 15) When the rRNA is not present in feces, Tr
is, 0.25% SD in EDTA buffer (pH 8-9)
Release from the cells by lysis for 10 min at room temperature in S and if feces are present.
Tris, 0.25 in EDTA buffer (pH 8-9)
2. A method according to claim 1, characterized in that the cells are released from the cells by lysis in % SDS at 75 DEG C. for 10 minutes, followed by filtration. 16) B. A solution for releasing rRNA from Campylobacter cells; B. A first labeled oligonucleotide that is complementary to Campylobacter rRNA and to which the label is bound to the first support. a second label, the probe is complementary to Campylobacter rRNA, the label is a detection molecule, and the first probe or the second probe is specific for a region of Campylobacter 16S rRNA; detecting and quantifying Campylobacter in a sample comprising an oligonucleotide probe; d. a solid support bound to a second support-binding partner that is complementary to the first support-binding partner; A test kit suitable for 17) complementary to Campylobacter 16S rRNA;
It is also specific and has a 5′-[gene sequence]-
An oligonucleotide probe having a 3' nucleotide chain. 18) complementary to Campylobacter 16S rRNA;
It is also specific and has a 5′-[gene sequence]-
An oligonucleotide probe having a 3' nucleotide chain. 19) complementary to Campylobacter 16S rRNA;
It is also specific and has a 5′-[gene sequence]-
An oligonucleotide probe having a 3' nucleotide chain. 20) complementary to Campylobacter 16S rRNA;
And it is specific, a, 5'-[there is a gene sequence] -3', or b, 5'-[there is a gene sequence] -3', or c, 5'-[there is a gene sequence] - 3', or d, 5'-[There is a gene sequence] -3', or e, 5'-[There is a gene sequence] -3', or f, 5'-[There is a gene sequence]-3' , or g, 5'-[gene sequence is available] -3', or h, 5'-[gene sequence is available] -3', or i, 5'-[gene sequence is available] -3', or j, 5'-[There is a gene sequence] -3', or k, 5'-[There is a gene sequence] -3', or l, 5'-[There is a gene sequence] -3', or m, 5'-[There is a gene sequence] An oligonucleotide probe consisting of a group of nucleotide chains consisting of -3'. 21) In a method for evaluating and analyzing the presence of one or more target nucleic acid chains in a sample, a. A sample consisting of cells of one or more cell types and estimated to contain one or more target nucleic acid chains; (b) releasing the target nucleic acid chain from the cells of the sample; (c) hybridizing the target nucleic acid chain, assuming it is present in the sample, with a plurality of different oligonucleotide probe units to generate a plurality of hybrid complexes. each probe unit comprises at least two labeled oligonucleotide probes, each complementary to a region of the target nucleic acid, and at least one of which is complementary to a region of the target nucleic acid. at least one of the probes of the unit has a nucleotide linkage specific for
labeling one or more first supports with a binding partner;
labeling at least one of the probes of the unit with one or more different detector molecules; d. capturing the hybrid complex on a solid support;
forming a sandwich composite, the solid support comprising:
having one or more second support-binding particles immobilized thereon that are complementary to the first support-binding partner; binds to a partner that binds to the second support, e. separates the sandwich complex from the sample and unhybridized probe of the unit, and f. binds to the sandwich complex. A method characterized in that the presence of one or more target nucleic acid chains is detected by activation of detector molecules, each of which gives a distinct activation reaction. 22) The method according to claim 21, wherein the partner that binds to the first support is a hapten, and the partner that binds to the second support is an antibody. 23) The method of claim 21, wherein the detection molecule is a chemiluminescent molecule. 24) The method of claim 23, wherein the chemiluminescent molecule is dimethylacridinium ester. 25) A method according to claim 21, characterized in that the solid support is a paramagnetic particle. 26) activating the chemiluminescent molecule by adding a first reagent solution and then, following an incubation period, adding a second reagent solution to initiate a detectable photoreaction; Reagent solution is 1.0N in 0.5% solution of H_2O_2
22. The method of claim 21, wherein HNO_3 and the second reagent solution is 2.5N NaOH in a 0.5% surfactant solution. 27) The surfactant is Arquad.
27. The method of claim 26. 28) The method of claim 21, wherein the chemiluminescent molecule is an acridinium ester. 29) the partner binding to the first support is biotin or avidin/strepavidin, respectively;
and the partner binding to the second support is biotin or avidin/strepavidin. 30) The method according to claim 22, wherein the partner that binds to the first support is dinitrophenol, and the partner that binds to the second support is an anti-dinitrophenol antibody. 31) The method according to claim 21, wherein the detection molecule is an enzyme. 32) A method according to claim 21, characterized in that the target nucleic acid chain consists of a 5S or 16S or 23S ribosomal unit of a bacterial genus or species. 33) One of the above units is a nucleotide chain a, 5'-[there is a gene sequence] -3'', or b, 5'-[there is a gene sequence] -3', or c, 5'-[there is a gene sequence ] -3', or d, 5'-[There is a gene sequence] -3', or e, 5'-[There is a gene sequence] -3', or f, 5'-[There is a gene sequence ]-3', or g, 5'-[There is a gene sequence]-3', or h,
5'-[There is a gene sequence] -3', or i, 5'-[There is a gene sequence] -3', or j, 5'-[There is a gene sequence] -3', or k, 5' - [There is a gene sequence] -3', or l, 5' - [There is a gene sequence] -3', or m, 5' - [There is a gene sequence] -3' At least two members of the group Consisting of members
The nucleotide chain is Campylobacter 16S rRNA
22. The method of claim 21, wherein the method is specific for and complementary to Campylobacter and it is believed that Campylobacter cells are present in the sample. 34) further comprising quantifying the number of bacterial cells of one or more genera or species in said sample, said test evaluation analysis comprising:
22. The method of claim 21, wherein approximately 1 x 10^4 bacterial cells can be quantified per milliliter of sample. 35) A method according to claim 21, characterized in that said different detector molecules are activated sequentially or simultaneously. 36) When the rRNA is not present in feces, Tr
is, 0.25% SD in EDTA buffer (pH 8-9)
Release from the cells by lysis for 10 min at room temperature in S and if feces are present.
is Tris, 0.25 in EDTA buffer (pH 8-9)
22. A method according to claim 21, characterized in that the cell is released from the cells by lysis in %SDS at 75[deg.]C for 10 minutes, followed by filtration. 37) B. A solution for releasing labeled nucleic acid chains from cells; B. At least two different solutions, each complementary to the target nucleic acid chain and to which the label is bound to the first support. (c) Each labeled oligonucleotide probe has a distinguishable label and is complementary to the target nucleic acid chain like the first probe, the label is a detection molecule, and the first probe or at least one of the second probes is complementary to the same target nucleic acid chain and is specific for a region of the target nucleic acid chain, at least two different second labeled oligonucleotide probes; D. one or more target nucleic acids in a sample consisting of a labeled solid support that is complementary to the first support and that is the second support. Test kit suitable for detecting and quantifying the presence of linkage. 38) The oligonucleotide probe is A., E. bases 0163-0214 of E. coli 16S rRNA, or bases 0176-0205 of coli 16S rRNA,
Or Ha, E. Base 0163-0 of coli 16S rRNA
204, or D.E. Base 04 of coli 16S rRNA
37-0463, or Ho, E. bases 0641-0671 of E. coli 16S rRNA, or E. Cori 16Sr
RNA bases 0821-0845, or E. bases 0195-0215 of coli 16S rRNA, or
E. bases 0156-0185 of E. coli 16S rRNA, or bases 0156-0185 of E. coli 16S rRNA. Base 0829-08 of coli 16S rRNA
54, or Nu, E. Base 110 of coli 16S rRNA
7-1140, or Le, E. 2. The method according to claim 1, which corresponds to a region consisting of bases 0706-0732 of E. coli 16S rRNA. 39) The oligonucleotide probe may be A., E. bases 0163-0214 of E. coli 16S rRNA, or bases 0176-0205 of coli 16S rRNA,
Or Ha, E. Base 0163-0 of coli 16S rRNA
204, or D.E. Base 04 of coli 16S rRNA
37-0463, or Ho, E. bases 0641-0671 of E. coli 16S rRNA, or E. Cori 16Sr
RNA bases 0821-0845, or E. bases 0195-0215 of coli 16S rRNA, or
E. bases 0156-0185 of E. coli 16S rRNA, or bases 0156-0185 of E. coli 16S rRNA. Base 0829-08 of coli 16S rRNA
54, or Nu, E. Base 110 of coli 16S rRNA
7-1140, or Le, E. 22. The method according to claim 21, wherein the method corresponds to a region consisting of bases 0706-0732 of E. coli 16S rRNA. 40) The oligonucleotide probe is A., E. bases 0163-0268 of E. coli 16S rRNA, or coli 16S rRNA bases 0391-0450,
Or Ha, E. Base 0631-0 of coli 16S rRNA
868, or d.E. Base 11 of coli 16S rRNA
2. The method according to claim 1, wherein the area corresponds to a region consisting of 02-1145. 41) The oligonucleotide probe comprises A., E. bases 0163-0268 of E. coli 16S rRNA, or coli 16S rRNA bases 0391-0450,
Or Ha, E. Base 0631-0 of coli 16S rRNA
868, or d.E. Base 11 of coli 16S rRNA
17. The method according to claim 16, characterized in that it corresponds to a region consisting of 02-1145. 42) Each of the probes contains Campylobacter 16
2. The method according to claim 1, wherein the method is specific and complementary to mutually exclusive regions of SrRNA. 43) Each of the probes contains Campylobacter 16
17. The method according to claim 16, wherein the method is specific and complementary to mutually exclusive regions of SrRNA. 44) The method of claim 21, wherein each of the probes is specific and complementary to a mutually exclusive region of the target nucleic acid chain. 45) The method of claim 37, wherein each of the probes is specific and complementary to a mutually exclusive region of the target nucleic acid chain.