JPH06502534A - Methods and kits for analyzing microorganisms - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Methods and kits for analyzing microorganisms The present invention relates to a method and kit for analyzing microorganisms in a sample.

Samples for clinical tests (e.g. urine, feces, saliva, sputum, serum, blood, biopsy samples, etc.) ) or food contains a small number of potentially pathogenic bacterial species among a large number of other microbial species. Can be mixed. These potentially pathogenic bacteria are analyzed using selective media.

For example, the typical method for analyzing Salmonella bacteria is Lumonella/Shigella (Shige Ia) culture on agar medium and Selena Biochemical and serological identification is performed after surface expansion on an agar medium rich in chromatin. Ru. This typical method has substantial drawbacks, especially the long and intensive labor required. There was a drawback.

U.S. Pat. No. 4,677,055 discloses a variation of the above exemplary method. The microorganisms to be analyzed are first isolated from the sample. to this method According to A magnetic gel to which an antibody against the determinant is bound is contacted.

Next, take out these magnetic particles with a magnetic rod and plant them on an agar medium with a cross section of about 1 cm. Allow spots to form. Place this agar medium on a colony of the same size as the spot above. After incubating so that a Bacteria in Ronnie can be identified.

Also, the presence of certain types of bacteria, such as Salmonella, in laboratory samples or food. Enzyme immunoassay using polyclonal or monoclonal antibodies or ELISA (enzyme-1 inked immunoso+ben+a A method of analysis using ssay) has also been developed. This kind of analysis will take no more than 1-2 days. However, before starting the actual analysis, the microorganisms in the sample are first grown in a growth medium. We have to increase it. Moreover, such immunochemical detection requires It is difficult to distinguish between antiserum and antiserum, and a sensitivity higher than 106 cells/mQ cannot be achieved. There is a drawback that there is no

The above-mentioned U.S. Pat. No. 4,677.055 also describes an immunological method for characterizing bacteria. The technology is also disclosed. For this purpose, place a well in the immediate vicinity of the colony on the agar plate. Prepare a chamber, fill it with specific antiserum, and lyse the colonies. from the colony Diffusion of antigen and antibody from the antiserum-filled cavity produces an immunoprecipitate; This precipitate is then stained. This operation requires approximately 2 days.

Instead, a new method for microbial analysis using modern DNA technology has been proposed. There is. Specific to a specific microorganism (specific genus, species or strain) in terms of speed and sensitivity of analysis PCR using specific primers (poly[oerxse chain rea Amplification of DNA sequences by cation) is considered to be promising. However, PCR technology There is a practical problem that amplification of the target DNA is inhibited by a large amount of mixed DNA. There are some drawbacks.

Jansen et al. (PNAS USA 87.1990.2867 -2871) discloses an analysis system for hepatitis A virus (HAV).

This analysis system uses 1 and 5 mH Etzbenodorf tubes as anti-HAV tubes. Place the ambassador sample coated with monoclonal antibody and containing HAV into a tube 1 After an overnight incubation, the suspension was washed away and an appropriate buffer was added to separate the RNA from ( (HAV is an RNA virus) performs reverse transcription into DNA and transfers this DNA to other Perform PCR in buffer solution. This operation takes approximately 24 hours. Advantages of this system As for the specificity at the antibody stage, the sensitivity of PCR and the specificity of PCR products, There may be a combination of confirmations. However, this method also has substantial disadvantages, That is, it has the disadvantage that it cannot be used for analysis of pathogenic bacteria such as Salmonella enterica. bacteria Unlike infections, the presence of closely related microbial species is rarely a problem in viral infections. Very little or not at all. Also, samples containing bacteria are suitable for PCR. It is practically impossible to return to such a state.

On the other hand, there are several protocols for PCR for single-strain culture fluids. for example, Bacteria were made into pellets by centrifugation, water was added, and the protocol was heated at 95°C for 10 minutes. - Add Lure K and incubate at 55°C for 10 minutes. The protocol was incubated at 95°C for 15 After inactivating by heating for 1 minute, add RNase and incubate at 37℃ for 15 minutes. start. Perform PCR for the first time after these operations [Barry et al. , BioItechnology 8, 1990, 233-236].

If this is the case, after bacteriolysis and treatment, first phenol extraction and In some cases, DNA purified by performing a cesium chloride gradient is used as a starting material. [Berne+ et al., J., Cl1n, Crobio 1.27.1989.2492-2496; Wilson et al. J, Cl1n, crobio1.28゜1990, 1942-+946; Riki Itis (p+1hay+1s) et al., J, Cl1n, Microbio l, 28.1990.1913-19171゜Similar to the above method A protocol for DNA purification using phenol extraction and cesium chloride gradient. Accordingly, there are methods for extracting viral DNA from biopsy samples, serum or blood [ Kaneko (Kaneko> et al., PNASυSA 116.1189, 312-3 16: Maitland et al., Br1t, J. Cance r 59.1989, 698-703.

Infection with intestinal bacteria (ente+obac+e+1aceae) occurs through feces. There may be very few particles and other bacteria present in the water. Samples can be prepared using a relatively simple method. i.e. two centrifugations or A filtration operation is sufficient. Next, remove the DNA from the obtained cells using a lysis buffer. These cells were treated with 50mM KCI, 10mM Tris-HCI. (pHa, t3), 1.5 mM MgCl2.0.01% gelatin, 0.05 H/ml proteinase K, 20mM dithiothreitol, and 1.8 Add μM SDS. After 15 seconds of voltenization, the mixture was heated to 37 °C for 1 Proteins were isolated by incubating for ~1.5 hours, then heating at 80°C for 5 minutes. Inactivate Nase K and perform PCR [Atlas and Bei ), Innis, Gelfand, Suni Edited by Sninsky and White, City of San Diego “PCR Protocols - Methods and Applications” published by Academic Press, Inc. Guide (PCR protocols; a guide to method s andappl 1 calions), 399-406].

In addition, several mixing and centrifugation operations were performed to isolate bacteria from the waste sample. This is followed by lysis and, in particular, DNA purification using a cesium chloride gradient. Selfan and Atlas, Appl, En ++ironm, Microbiol, 54°1988, 2185-219 11° The present invention, unlike all prior art, allows for the analysis of bacteria, fungi and yeasts in various samples. Providing methods and kits that enable rapid detection in a specific and sensitive manner do.

According to the first aspect of the present invention, there is provided a method for analyzing microorganisms in a sample. using antibodies with specific affinity for microorganisms and solid supports for these antibodies. These microorganisms are isolated from the sample, and then the DNA of the isolated microorganisms is analyzed. A method that includes PCR using primers specific to the microorganism to be used. law is provided.

According to the second aspect of the present invention, an antimicrobial agent having specific affinity for the microorganism to be detected is provided. A kit for the analysis of microorganisms in samples, including human bodies, in which the antibodies are attached to their solid support. PCR primers that bind or are specific to the microorganism to be analyzed In addition, the antibody may be attached to a solid support provided in the kit. A kit is provided, characterized in that the kit is provided with reagents capable of.

The method of the invention is particularly applicable to microorganisms consisting of bacteria, fungi or yeasts of a particular genus, species or strain. Suitable for analyzing things. These microorganisms to be detected are isolated from the sample. The use of antibodies with specific affinity for these microorganisms and the D PCR primers that amplify NA and are specific to the microorganisms to be analyzed. In combination with the use of Even different strains can be distinguished.

The present invention applies to samples used in clinical tests (urine, feces, sputum, saliva, serum, blood, biopsy samples). etc.) or methods for analyzing pathogens in food, such as bread, beer, Yeast and/or fungi used in the production of consumable products such as wine, cheese, yogurt, etc. It also includes identification methods. However, one aspect of the present invention is that conventional methods cannot It is preferable to use it for analysis of pathogenic bacteria that could not be analyzed. For example, the present invention specifically supports Lumonella, Klebsiella or Listeria It is suitable for the analysis of microorganisms consisting of bacteria of one genus within the genus Ria). For one thing Since bacterial analysis is a preferred application of the present invention, detailed descriptions are often given hereafter for convenience. Although described with reference to bacteria, the present invention is not limited thereto.

For the most reliable results, the antibody (preferably monoclonal antibodies) can react with living bacteria; It is necessary to have specific affinity for and be able to bind to bacteria. Especially containing DNA. Since it is a living bacterium, the final detection in the present invention is based on the It is carried out using bacterial DNA.

According to the invention, therefore, in order to isolate the microorganisms to be analyzed from a sample, it is possible to Using a monoclonal antibody produced against the microorganism that exists, that is, using the antibody against the microorganism. The test animals (usually rats) that produce the bacteria are exposed to living bacteria (at least under conditions close to these). It is preferable that the immunization is carried out using a potato. Infects humans but not rats For viruses like HAV, which are not pathogenic, this is less of a problem. Although this is not a problem, in the case of Salsonella bacteria that are also pathogenic to mice, Such immunization is considered impossible.

The only monoclonal antibodies against Salmonella that have been published so far are killed bacteria. The sample containing this bacterium must first be #,s reacted with. It means something.

This generally applies to all immunoassays currently used for the detection of Salmonella. This applies to Sei. However, boiling the bacteria lyses the cells and removes the DNA. Since it disappears, PCR is no longer possible.

Therefore, when trying to obtain monoclonal antibodies against living pathogens, Conventional immunization methods cannot be used. There are two obvious reasons for this.

First, there is a method of using an adjuvant to obtain an immune response; In this case, the native proteins of the organisms in the adjuvant are denatured and many epitopes are destroyed. It has the disadvantage of being Therefore, treatment with adjuvants or adjuvants A monoclonal antibody that reacts with protein sites whose structure does not change even after internalization. [Luk et al., J. Immunol, Met. h, +29.1990.243-250; Mason Smith 1th) and Botter (Po+16r), J. Immunol, 114.　 +975. ll1f47-1850; Feng et al., J. Gen , Microbiol, 136.1990.337-342.

Second, in the case of a natural organism that is pathogenic to the animal to be immunized, immunization is basically impossible.

To avoid this problem, the biological protein is often used in isolated form during actual immunization. Often used, but because carefully isolated proteins are injected, usually at the same time as an adjuvant show only a very weak immune response [Sadallah et al., J. Infect, Dis.

+61.1990.59-64]. Complete denaturation of organisms or isolated proteins As a result, the antigen becomes more immunogenic, but treatment with an adjuvant Many three-dimensional structural epitopes are missing due to chemical or other denaturation methods. cormorant. Therefore, the stimulated immune system will not mount any immune response against such epitopes. Not shown. However, on living bacteria, epitopes in the three-dimensional structure are There are particularly many issues related to supply and movement.

According to the present invention, several solutions to the above-mentioned problems have been discovered. for natural epitopes In order to obtain an immune response under these conditions, the present invention uses live bacteria or the closest organism Perform immunization using bacteria. One method involves introducing bacteria into laboratory animals along with antibiotics. Inject. As a result, the resulting bacteriostatic effect (bac+eriosta+ic e ffec+), the growth of which is inhibited, but the bacteria that are still alive can invade the immune system. Become so. According to the second method for obtaining an immune response against living bacteria , the strain is treated with a low concentration of formalin. A possible outcome is that by phagocytic cells. Bacterial engulfment (pathogens are often taken up by phagocytes and presented intracellularly to the immune system. (continue to survive invisibly), but the degree of inhibition depends on the immune system of the bacterium. The level is such that invasion into cells occurs.

Therefore, according to the present invention, for the production of the above-mentioned monoclonal antibodies, bacterial growth is Immunization using the live microorganisms described above in combination with an amount of antibiotic that inhibits growth. The antibiotic is used in an amount that inhibits bacterial growth or inhibits the growth of the living microorganism. Immunization can be carried out using microorganisms obtained by processing living organisms, or by using the antibiotics or food. live with formaldehyde in amounts that inhibit the uptake of bacteria by cells It is preferable to perform immunization using a microorganism obtained by treating the microorganism.

According to the invention, the antibodies used to isolate the microorganism to be analyzed are bound to a solid support. (or, in some cases, The body and solid support are coated with biotin/avidium to allow for subsequent camping. If it has been appropriately modified by or after reaction with microorganisms present in the material). The properties of the solid supports mentioned above are metals that can be separated from the rest of the sample by, for example, centrifugation. , preferably consisting of inert particles of plastic or glass, according to the invention. For example, it is possible to use magnetic particles, which are very easy to pick up from a sample using magnetic force. Very preferable. Compared to methods that use the wall of the tube as a solid support for the antibody, When particles (particularly magnetic particles) are used, proper contact with the sample can prevent microorganisms. It takes very little time to achieve optimal union with the body.

Therefore, according to the present invention, by using magnetic particles as a solid support for the antibody, It is highly preferred to isolate the microorganisms to be analyzed from the sample. Also, analysis Magnetic particles bound to monoclonal antibodies that have specific affinity for the target microorganism microorganisms are added to the sample, and after incubation, the microorganisms to be analyzed are added to the sample. The analysis is carried out by separating those microorganisms bound to the magnetic particles from the magnetic particles. Most preferably, the microorganism of interest is isolated from the sample. Furthermore, the monochrome Magnetic particles that have specific affinity for the monoclonal antibody are coated with the monoclonal antibody. obtained by incubating the monoclonal antibody with the magnetic particles, The monoclonal antibody binds and has a specific affinity for the microorganism to be analyzed. It is preferable to use magnetic particles having the following structure. The monoclonal antibody In the case of immunoglobulins of murine origin, the magnetic particles are e.g. It is coated with goat immunoglobulin, which has specificity for murine immunoglobulin. good.

Bacteria and other microorganisms exhibit specific binding or adhesion effects to solid surfaces. . This means that the sample to be examined may contain other bacterial species in addition to the Salmonella and other microorganisms to be analyzed. This becomes a particularly big problem when it also contains. This problem should be analyzed by a few If the bacteria is present in excess among other microorganisms in the mosquito, e.g. 1 million Escherichia coli (E. If one Salmonella is present for sche + 1 cia coli), becomes a serious problem. In such cases, the method disclosed in U.S. Pat. No. 4,677.055 With this method, there is a complete excess of E. coli attached to the particles compared to Salmonella. Therefore, it would be insufficient. The present invention achieves the above by using PCR as a detection method. This completely overcomes the problems mentioned above. Appropriate selection of primers increases specificity and Although high sensitivity is ensured, obtaining these desirable properties requires P obtained by separating, lysing (for example by heating to 60-100°C) and centrifuging PCR is performed using the DNA released from the bacterial cells obtained as the supernatant for CR. is appropriate.

Therefore, according to the present invention, DNA can be separated from microorganisms bound to the magnetic particles. , it is preferred to subject the isolated DNA to PCH in the absence of the magnetic particles. So In addition, the present invention requires only a small amount of sample to be collected for PCR, and Even when the purpose is to analyze Listeria monocytogenes, etc., lysis buffer, phenol extraction and and cesium chloride gradients may not be required.

The PCR conditions themselves are similar to those often used in PCR such as Taq polymerase. The types of suitable DNA polymerases are well known to those skilled in the art (e.g. (see U.S. Pat. No. 4,683,202). However, regarding the selection of primers are specific to the method of the invention, and this selection makes the method of the invention specific to a particular genus. , may be specific for a particular species of microorganism, or even for a particular strain of microorganism. become certain. However, finding the right primer is not always easy.

For example, to ensure specificity for a particular genus of microorganisms, such as Salmonella. In other words, while identifying all strains and strains belonging to Salmonella by the method of the present invention, Primer selection becomes an issue when it is desired not to discriminate against bacteria of other genera.

It is known that rRNA sequences are very similar in certain genera; This has already been used in hybridization experiments (EP-A -0357306° See EP-A-0277237 and WO28103957 ). The use of rRNA primers was previously published by Chen et al. There is (FEMS Microb, Let+, 57.1989, 19-24 ), which is used in routine microbial analysis.

The use of rRNA primers is one possibility encompassed by the invention, but in all cases This does not mean that it is suitable for all cases. Especially in the case of Salmonella, the rRNA sequence It is known that there are significant sequence variations. Appropriate sequences to replace rRNA are Found in the sequence of the “DNA replication origin” (oriC) of bacterial chromosomes. The oriC sequence is known for several intestinal bacteria [Corn Barb ( Kornberg), Freeman of San Francisco and =+ +, (F [Supplementary information on DNA reproduction (S) published by [eeman and Co.] upplement to DNA + replication)” this or The iC sequence consists of a region conserved among intestinal bacteria and a mutable partial sequence. deer However, these mutable partial sequences are conserved internally in Salmonella genus etc. Therefore, it is unclear whether they can serve as the basis for PCR primers specific to bacterial genus. It was bright.

Therefore, the present invention provides primers based on partial sequences of rRNA sequences in PCR. This includes the use of Such an approach has actually been applied to Listeria monocytogenes has been found to be useful.

The present invention also provides a method for PCR based on a partial sequence of a DNA replication origin sequence. This includes using primers.

The first specific example of the above method is for the analysis of Salmonella bacteria, and the following original There is a method characterized in that a oligonucleotide primer is used in PCR. .

Primer 1: 5'-TTATTAGGATCGCGCCAGGC-3° Primer 2: 5'-AAAGAATAACCGTTGTTCAC-3' The use of the above primers of the present invention will be described in further detail in specific examples. Ru.

A second specific example of the above method is for the analysis of Klebsiella bacteria, and includes the following options: There is a method characterized in that a oligonucleotide primer is used in PCR. Ru.

Primer 1: 5'-CTTGTCTTGTGGATAAGTCA-3' Primer 2: 5°-TCTTCTGTGGATAACTATGC-3' This primer combination and Klebsiella specific monoclonal antibody Use has shown that the method of the invention is selective for Klebsiella bacteria. It has been proven through experimentation. This means that the Klebsiella oxyto force (Kleb siella oxy+oca) and Klebsiella pneumoniae (Klebs iella pneumoniae), the result was positive, and Escherich Esche+1chia coli, Citrobacter floi Cittobacter ireundii, Serratia marse Serraia marcescens), Proteus and Mira Squirrel (Pr.

teus m1rabilis), Enterobacter cloake (En4e+ obaever C1oacae), and Enterobacter xogenes (E le + obacter aerogenes), the result must be negative. means.

However, not all primers are useful and active in PCR. . Therefore, the following primers based on the oric sequence were used for the analysis of Klebsiella bacteria. A combination of these has been established.

Primer 1: 5'-CTTGTCTTGTGGATAAGTCA-3' Primer 2: 5'-AAGTATAACCGTTGCCTGA-3゜ In the examples below, we will use a substance that can bind to live Salmonella bacteria. A clonal antibody was used. Hybrids that produce these monoclonal antibodies The market was deposited with the ECACC (UK) on November 28, 1990 under the following deposit number: European Co11ection o[Anim@l Ce1l Cu 1tures) + This deposited: MAB 55-23-B7: ECACC90112807MAB 7O-2 -2A: ECACC90112808MAB 7l-40-Al: EC ACC90112809X production five Using PCR technology, a 160 bp partial sequence, which is the sequence of the origin of DNA replication, is increased. I did the width. For this purpose, Salmonella typhimurium Primer having a sequence derived from the origin of replication of lla [phimurium] - was used. These oligonucleotide primers were derived from Fermacia elke -Be Gene Assembler Plus Synthesizer (pharmacia) LKB Gene Assembler Plus 5ynthesixer) Synthesized by phosphoramidile technology using did.

After deprotection and excision from the solid support, the synthetic oligonucleotides were labeled with NAP Purified by 10 column chromatography. The obtained primers are as follows. ``Primer 1 + 5'-TTATTAGGATC'GCGC'' had the sequence CAGGC-3″ Primer 2: 5'-AAAGAATAACCGTTGTTCAC-3' Of the above primer set, 27 strains of Salmonella and other strains other than Salmonella Specificity for 19 strains of enterobacteriaceae was examined by PCR (Table A). PCR mixture ( Final volume: 100 pQ) is 10 mM Tr i 5-HC (1 (pH 8,3) , 50m M K CQ, 1, 5 m M M g CQ 2.0.001% (W/■) Gelatin, 100 μM of each dNTP, 0.5 μM of each primer, and and 1.25 U of Amp1i-Taq polymerase (California, USA). (manufactured by Cetus, Inc., Emeryville, USA) was used. The amplification is Perkin-Elmer Thermocycle (Perkin-Elmer The+m ocycle; Perkin-Elma, Norwalk, Connecticut, USA (manufactured by Instruments Inc.) for 25 cycles. One cycle is 9 1 minute denaturation step at 4°C, 1 minute primer annealing step at 50°C, and 1 minute primer annealing step at 72°C. It consisted of a 1 second extension step. After amplification, 15μQ of sample was transferred to 1.5% agarose. The electrophoresed DNA was subjected to gel electrophoresis and stained with ethidium bromide. Like NTP, it was obtained from Beilinger Mannheim, Germany).

After amplification, DNA fragments of the desired length are isolated not only from Salmonella typhimurium but also from Salmonella typhimurium. , was found for all Salmonella bacteria examined. On the other hand, Shigella, Escherichia ・Other intestinal bacteria such as Coli, Citrobacter, and Pseudomonas are amplified. No fragments were found. Therefore, the selected primers are specific to Salmonella It was determined that this was the target.

Selectivity and specificity of the method of the invention Salmonella typhimi serially diluted to 5X10' to 5X102 cells/mQ Lium (100 μQ each) was investigated using the method of the present invention. As magnetic particles, rice MagniSort (obtained from DuPont, Ilmington, Praue State) Magnisotl M particles (magnetic chromium dioxide) were used. Also these The particles are coated with mouse immunoglobulin specific for IgG and IgM. Ta. Magnetic particles of monoclonal antibody (SUBCL, KIgM) for salmonella bacteria Coupling was performed by co-coating in phosphate-buffered saline (gPBs) containing 1% gelatin. Incubate the hybridoma culture supernatant with the magnetic particles at 35°C for 5 minutes. It happened by doing. Magnetic particles were isolated using a magnet after discarding the supernatant.

Samples for analysis were suspended in gPBs and magnetic particles were added. Incubate for 15 minutes at 35℃ After heating, the magnetic particles were separated using a magnet and washed three times with gPBs.

After the final washing operation, the magnetic particles were resuspended in double distilled water.

In an attempt to perform direct PCR on bacteria extracted with magnetic particles, amplification was was found to be inhibited by sexual particles. For this reason, the sample was heated to 100℃ for 5 minutes. The bacterial bodies are disrupted by incubation, and after a brief centrifugation, the bacteria are The supernatant fraction containing DNA was subjected to PCR. PCR has 35 DNA amplification cycles It was carried out according to the method described above.

As a result (not shown here), 103 cells could be detected using the method of the present invention. I found out that it can be done. In addition, magnetic particles bound to Salmonella and monoclonal antibodies The supernatant obtained after bacterial culture was also subjected to PCR to measure the binding ability with No amplification of the target DNA was observed. From this, salmonella and magnetic particles It is concluded that the binding with the above monoclonal antibody is complete.

Subsequent experiments showed that according to the method of the present invention, 107 bacteria other than Salmonella were detected. Bacteria [Escherichia coli, Pseudomonas aeruginosa] aeruginosa), Klebsiella oxytophora and Citrobacter -・Ability to analyze 103 Salmonella bacteria in samples containing I.D. was shown. Escherichia coli, Pseudomonas aeruginosa, Klebsi Construct using Citrobacter oxytoxin and Citrobacter freundii and gPBS. No detectable amplification was observed in the troll experiments.

Table A PCR amplification of Salmonella and other intestinal bacteria PCR of Salmonella Amplification serotype S, Durazzo (S, Durazzo) 10AS, Clinical Isolation ToM1A (S, clinical 1solate M) S, Typhimurium L +A (S, +yphimurium L) S, Typhimurium 1724 10 B (S, +7phimutium + 724) PCR Salmonella Amplification serotype S, Typhimurium 14H+B (S, [phimueium 14) 1) S, bted = - (S, bted enY) 10BS, Derby (S, derby) 10BS, Heidelberg 10 B (S, heidelbe+g) S, Prandenburg 10B (S, brandenbu+g) S, clinical isolate 10.2 + B (S, clinical 1so late 10.2) S, St. Paul + B (S, 5ain+paul) S, baratifi B + B (S, pa+a+yphi B) S, Aborta Suiki 10B (S, abortus equi) S, Infantis (S, 1nfan! is) + CIS, Newborn To (S, newpo++) 10C2S, Belem (S, belem) + C5, Clinical isolate 1159 + C1 (S, clinical 1sol ate 859) S, baratifi C (S, pa+atypbi C) 10C 5, Pana? (S, panam@) + DS, Typhosa (5,17Ph osa) + DS, Enteriditis + D (S. enleridilis) PCR Salmonella Amplification serotype S, shalgar = (S, sha+gani) + ES, give (S, give ) + E S, Pretoria (S, p+eto+ia) 10FS, Atlanta (S, atla n+a) 10GS, Worthington (S, vor+hinglon) + GE Sherihia Cori - (Escherichia coli) Escherichia coli 07KI - (Escherichia coli 07Kl) (Klebsiella p pneumonia 1.88) Klebsiella oxytoforce - (Klebsiella oxyjoca) Citrobacter freundii − (Citrobacter order) Citrobacter diapersus − (Citrobacter diversus) Serratia riffuscens − (Serralia l1qufaciens) Serratia marcescens - (Serralia 1lIlarcescens) Enterobacter agro Melance - (Er++e+obac+er agglome+ans) PCR of salmonella Amplification serotype Enterobacter erogenes (Enterobac+e+ aetogenes) (Enterobac+e + cloaceae) Proteus mirabilis - (Proteus mi+abilis) (Proteus vulgaris) ) International search report international search report NL　9100230 S^　53610 Continuation of front page (81) Specified times EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, NL, SE), C A, JP, US

Claims (22)

[Claims] 1. A method for analyzing symptomatic organisms in a sample that has a specific affinity for the microorganisms to be detected. These microorganisms are isolated from the sample using antibodies and solid supports for these antibodies. The DNA of the isolated microorganism is then isolated in a manner specific to the microorganism to be analyzed. A method comprising subjecting to PCR using primers. 2. The microorganism to be analyzed is a specific genus, species, or strain of bacteria, fungi, or yeast. The method according to claim 1, characterized in that: 3. If the above microorganism to be analyzed is Salmonella, Klebsiella or Listeria spp. The method according to claim 1, characterized in that it consists of bacteria of one genus of bacteria. 4. In order to isolate the above-mentioned microorganisms to be analyzed from the sample, the living microorganisms are according to claim 1, characterized in that a monoclonal antibody produced against the Method. 5. For the production of the above monoclonal antibodies, use an amount of antibiotic that inhibits bacterial growth. Immunization can be carried out using the above-mentioned living microorganisms in combination with substances, or Microorganisms obtained by treating living microorganisms with an amount of the antibiotic that inhibits their growth. Immunization is carried out using a living organism, or an amount of protein that inhibits the uptake of bacteria by phagocytes is used. Using a microorganism obtained by treating the living microorganism with lumaldehyde, The method according to claim 4, characterized in that immunization is performed. 6. By using magnetic particles as a solid support for the antibody, the microorganism to be analyzed is A method according to claim 1, characterized in that the substance is isolated from the sample. 7. A monoclonal antibody with specific affinity for the microorganism to be analyzed binds to it. Add magnetic particles to the sample and, after incubation, confirm that the microorganism to be analyzed is present. by separating those microorganisms bound to the magnetic particles from the magnetic particles. , wherein the microorganism to be analyzed is isolated from the sample. Method. 8. Magnetic particles having specific affinity for the monoclonal antibody are attached to the monoclonal antibody. the monoclonal antibody and incubating the monoclonal antibody with the magnetic particles. The monoclonal antibody obtained by Claim 7, characterized in that the magnetic particles are used to have magnetic affinity. the method of. 9. Isolating DNA from a microorganism bound to the magnetic particles and detecting the absence of the magnetic particles. The method according to claim 1, characterized in that the DNA isolated below is subjected to PCR. 10. In PCR, primers based on partial sequences of rRNA sequences can be used. The method according to claim 1, characterized in that: 11. In PCR, primers based on a partial sequence of the DNA replication origin sequence The method according to claim 1, characterized in that the method uses: 12. For the analysis of Salmonella bacteria, the following oligonucleotide primers are used: The method according to claim 1, characterized in that the method is used in PCR. Primer 1: [There is a sequence] Primer 2: [Sequence available] 13. For the analysis of Klebsiella bacteria, the following oligonucleotide primers are used: The method according to claim 1, characterized in that - is used in PCR. Primer 1: [There is a sequence] Primer 2: [Sequence available] 14. microorganisms in the sample, including antibodies that have specific affinity for the microorganism to be detected; in a kit for biological analysis, the antibodies are bound to their solid support, or , as well as a set of PCR primers specific to the microorganism to be analyzed. Provided with reagents capable of binding to the solid support for the antibody provided at the site. A kit characterized in that it is provided. 15. A specific affinity for the living microorganism to be analyzed. 15. The kit according to claim 14, comprising a monoclonal antibody having a specificity. 16. The above-mentioned live microorganisms are treated in combination with an amount of antibiotic that inhibits the growth of the bacteria. Immunization is carried out using a living organism, or an amount of the antibiotic is used to inhibit the growth of bacteria. immunization using the microorganisms obtained by treating the microorganisms, or using formaldehyde in an amount that inhibits bacterial uptake by phagocytes or phagocytes. It is produced by performing immunization using microorganisms obtained by processing living microorganisms. The kit according to claim 15, comprising the produced monoclonal antibody. 17. Claim 14, characterized in that it comprises magnetic particles as a solid support for the antibody. The kit described in. 18. Magnetic particles having specific affinity for the monoclonal antibody are attached to the monoclonal antibody. coat the monoclonal antibody with the magnetic particles, and incubate the monoclonal antibody with the magnetic particles. The monoclonal antibody obtained by Claim 17, characterized in that it contains magnetic particles having different affinities. Kit included. 19. It is characterized by containing a PCR primer based on a partial sequence of the rRNA sequence. The kit according to claim 14. 20. Contains PCR primers based on a partial sequence of the DNA replication origin sequence. The kit according to claim 14, characterized in that: 21. It is for the isolation of bacteria of the genus Salmonella, and the following oligonucleotide blocks for PCR are used. 15. The kit according to claim 14, comprising a primer. Primer 1: [There is a sequence] Primer 2: [Sequence available] 22. For analysis of Klebsiella bacteria, the following oligonucleotides for PCR are used. 15. The kit according to claim 14, comprising a primer. Primer 1: [Sequence available] Primer 2: [Sequence available] Details of the invention detailed explanation