WO1992008805A1

WO1992008805A1 - Method and kit for demonstrating microorganisms

Info

Publication number: WO1992008805A1
Application number: PCT/NL1991/000230
Authority: WO
Inventors: Jan Verhoef; Adriaan Camille Fluit; Ruurd Torensma
Original assignee: U-Gene Research B.V.
Priority date: 1990-11-15
Filing date: 1991-11-15
Publication date: 1992-05-29
Also published as: NL9002696A; JPH06502534A; EP0630413A1; CA2095843A1

Abstract

Detection of microorganisms by first isolating them from the sample by means of antibodies and a solid support for the antibodies and then, optionally after isolation of the DNA from the bound microorganisms, carrying out a PCR. The antibodies to be used are specific for the microorganisms to be detected and most preferably generated against still living microorganisms. Especially magnetic particles are suitable as solid support. In PCR, primers are used that are specific for the microorganisms to be detected, most preferably based on an rRNA sequence or a DNA replication origin sequence.

Description

Title: Method and kit for demonstrating microorganisms

This invention relates to a method and a kit for

demonstrating microorganisms in a sample.

In clinical samples (suchs as urine, feces, saliva, sputum, serum, blood, biopten and the like) or in foods potentially pathogenic species of bacteria may be present in small numbers among large numbers of contaminating other species of microorganisms. These potentially pathogenic bacteria are demonstrated by using selective media. Thus, the classical method of demonstrating Salmonella consists in a culture on Salmonella/Shigella agar and enrichment in selenite agar, followed by a biochemical and seriological

identification. This classical method has substantial

deficiencies, especially the fact that it requires much time and is labor-intensive.

U.S. patent 4,677,055 discloses a variant of this

classical method wherein the microorganisms to be determined are first isolated from the sample. According to this variant a biological medium in which pathogenic microorganisms with a specific antigenic determinant are present is contacted with a magnetic gel to which antibodies directed against this

determinant are coupled. The magnetic particles are then picked up with a magnetic rod and inoculated on an agar plate to form an imprint of about 1 cm in cross-section. The agar plate is incubated so that colonies (as large as the imprint) can be formed. Then, e.g., a classical bacteriological method can be used to identify the bacteria in the colony.

In the meantime, there have also been developed methods of determination wherein the presence of a certain type of bacteria, e.g., Salmonella, in clinical samples or foods is demonstrated with an enzyme immunoassay or an enzyme-linked immunosorbent assay (ELISA) using polyclonal or monoclonal antibodies. Such determinations take one or two days less, but yet the samples to be examined must first be enriched in a growth medium before the actual assay can be carried out.

Besides, the immunochemical detection has the further drawbacks that it is hard to distinguish related microorganisms with antisera and that no better sensitivity than 10⁶ cells/ml can be realized.

The above U.S. patent 4,677,055 also discloses an

immunological technique of determining the identity of the bacteria. For this purpose a recess is made in the agar at a short distance from the colonies, which recess is filled with a specific antiserum. The colonie are lysed. Diffusion of antigen from the colony and antibody from the recess filled with antiserum leads to an immune precipitate which may then be stained. This procedure takes about two days.

An alternative new approach for detecting microorganisms is offered by using modern DNA technology. As regards velocity and sensitivity, amplification of DNA sequences with PCR

(polymerase chain reaction) using primers specific for a certain microorganism (a certain genus, species or strain) seems to be a promising possibility. A substantial drawback of the PCR technique is, however, that the amplification of the target DNA sequence is inhibited by large amounts of

contaminating DNA.

Jansen et al, PNAS USA 87, 1990, 2867-2871, discloses a system for demonstrating the hepatitis A virus (HAV) wherein a 1.5 ml Eppendorf tube is coated with a monoclonal antibody directed against the hepatitis A virus. A feces sample that may contain HAV is placed in the tube, and after a one-night incubation the suspension is washed away. Addition of a suitable buffer is followed by reverse transcription to convert HAV RNA (HAV is an RNA virus) into DNA, which is then subjected to a PCR in another buffer. This procedure takes about 24 hours. Its advantage resides in the combination of specificity of the antibody step, sensitivity of PCR and simple confirmation of the specificity of the PCR products. However, this method also has substantial drawbacks, e.g., the fact that it cannot be used to demonstrate pathogenic

microorganisms such as Salmonella bacteria. In viral

infections, unlike in bacterial infections, the problem of closely related species of microorganisms being present seldom or never occurs. Recovery procedures for bacterially

contaminated samples to make them fit for a PCR are

practically not available.

On the other hand, a number of protocols are described for PCR on pure cultures. The bacteria are pelleted in a centrifuge, included in water and heated for 10 min at 95°C, followed by addition of proteinase K and incubation for 10 min at 55°C. The proteinase is deactivated within 15 min at 95°C, after which RNase is added and the incubation is continued for 15 min at 37°C. Only then is the PCR reaction carried out (Barry et al, Biotechnology 8, 1990, 233-236).

In some cases the starting material is even DNA which, after lysis and proteinase K treatment, is first purified via phenol extractions and optionally CsCl gradients (Bernet et al, J. Clin. Microbiol. 27, 1989, 2492-2496; Wilson et al, J. Clin. Microbiol. 28, 1990, 1942-1946; Plikaytis et al, J.

Clin. Microbiol. 28, 1990, 1913-1917).

Extraction of viral DNA from biopten, serum or blood proceeds via protocols comparable to the above method wherein DNA purification occurs via phenol extractions and CsCl gradients (see Kaneko et al, PNAS USA 86, 1989, 312-316;

Maitland et al, Brit. J. Cancer 59, 1989, 698-703).

In water in which fecal infection (Enterobacteriaπgap, may have occurred the sample can be worked up in a relatively easy manner because few particles and other bacteria are present. Two centrifugation steps or a filtration step are therefore sufficient. Subsequently, DNA is released from the resulting cells by means of lysis buffer. To the resulting cells are added 50 mM KCl, 10 mM Tris-HCl (pH 8.13), 1.5 mM MgCl₂, 0.01% gelatin, 0.05 mg/ml proteinase K, 20 mM

dithiothreitol, and 1.8 μM SDS. After 15 sec vortexes the mixture is incubated for l to 1.5 h at 37°C, then the

proteinase K is deactivated by 5 min heating to 80°C.

Subsequently, PCR is carried out (see Atlas and Bej, in "PCR protocols; a guide to methods and applications", Innis, Gelfand, Sninsky and White eds, Academic Press, San Diego, 399-406).

For the isolation of bacteria from soil samples a number of blending and centrifugation steps are used, followed by a lysis step and DNA purification via, among other things, a CsCl gradient (Steffan and Atlas, Appl. Environm. Microbiol. 54, 1988, 2185-2191).

The invention provides a method and a kit which, unlike all the previously proposed techniques, enable microorganisms such as bacteria, fungi and yeasts to be demonstrated in different kinds of samples in a rapid, specific and sensitive manner.

The invention relates in a first aspect to a method for demonstrating microorganisms in a sample wherein the

microorganisms to be detected are isolated from the sample by means of antibodies having a specific affinity for these microorganisms and a solid support for these antibodies, and then subjecting the DNA of the microorganisms thus isolated to a PCR in which primers specific for the microorganisms to be demonstrated are used.

In a second aspect the invention relates to a kit for demonstrating microorganisms in a sample, comprising

antibodies having a specific affinity for the microorganisms to be detected, which antibodies are bound to a solid support for these antibodies or are provided with an agent by means of which they can be bound to a solid support for the antibodies likewise belonging to the kit, as well as a set of primers for a PCR that are specific for the microorganisms to be

demonstrated.

The method according to the invention is particularly suited for demonstrating microorganisms consisting of

bacteria, fungi or yeasts of a certain genus, a certain species, or a certain strain, and the combination of the use of antibodies having a specific affinity for the

microorganisms to be detected so as to isolate these very microorganisms from the sample and the use of a set of primers for a PCR that are specific for the microorganisms to be demonstrated so as to amplify the very DNA of these

microorganisms ensures that, e.g., in bacteria the method can distinguish between different genera or between different species, or even between different strains.

The invention is not limited to methods for demonstrating pathogenic bacteria in clinical samples (suchs as urine, feces, sputum, saliva, serum, blood, biopten and the like) or foods but also comprises methods that enable identification of, e.g., the yeasts and/oder fungi used for the preparation of consumable products such as bread, beer, wine, cheese, yoghurt and the like. Preferably, however, the invention is utilized to demonstrate pathogenic bacteria, which is not possible with the prevailing techniques in a satisfactory manner. Thus, the invention is particularly suited for demonstrating microorganisms consisting of bacteria of one of the genera Salmonella, Klebsiella or Listeria. Partly in view of the preference for the use of the invention to determine bacteria the further description of the invention will often refer to bacteria, for the sake of convenience, without contemplating to limit the invention in this respect.

For a result to be optimally reliable it is required that the antibodies to be used for the isolation of the

contemplated microorganisms (preferably, monoclonal

antibodies) are capable of reacting with still living

bacteria, i.e. have a specific affinity for and can bind to the still living bacteria. For it is particularly the still living bacteria that contain DNA, and in the method of the invention the final detection occurs by means of the bacterial DNA.

According to the invention it is therefore preferred that for the isolation of the microorganisms to be demonstrated from the sample monoclonal antibodies are used which have been generated against the living microorganism, i.e. that the test animal used as source for the antibodies (mostly a mouse) has been immunized with the living bacteria (at least bacteria closely approaching this condition). In the case of human viruses that are not pathogenic to the mouse, such as HAV, this is not too great a problem, but in the case of, e.g.,

Salmonella bacteria that are also pathogenic to the mouse, such an immunization does not seem possible.

All hitherto described monoclonal antibodies against

Salmonella react with dead bacteria only, which means that the sample containing the bacteria must be boiled first. This generally applies to all presently available Immunological assays for the detection of Salmonella. Boiling these

bacteria, however, results in that they lyse and lose their DNA contents. Then PCR is of course no longer possible.

Therefore, the prevailing methods of immunization cannot be used if monoclonal antibodies against still living

pathogenic bacteria are to be obtained. Two obvious reasons can be indicated for this. Firstly, adjuvants are used to obtain an immune response. These adjuvants, however, have the drawback that the native proteins of the organism in the adjuvant denature so that many epitopes are destroyed.

Therefore, especially monoclonal antibodies are thus obtained which react with portions of the proteins which do not change their structure after treatment with or incorporation in an adjuvant (see Luk et al, J. Immunol. Meth. 129, 1990, 243-250; Mason Smith and Potter, J. Immunol. 114, 1975, 1847-1850; Feng et al, J. Gen. Microbiol. 136, 1990, 337-342).

Secondly, immunization with native organisms is basically impossible in cases that the organism is pathogenic to the animal to be immunized. To avoid this problem, proteins of the organism in isolated form are often used in practice for the immunization. The proteins, however, generate a very weak immune response, for which reason the carefully isolated proteins are mostly injected simultaneously with an adjuvant (see Sadallah et al, J. Infect. Dis. 161, 1990, 59-64). As a result of complete denaturation of the organism or the isolated proteins the antigens become much more immunogenic. However, owing to the treatments with adjuvants or other methods of denaturation many conformation epitopes are lost. The immune system thus stimulated will therefore give no immune response against such conformation epitopes. However, on living bacteria especially conformation epitopes occur because these attend to food supply and movement of the organism.

According to the invention several solutions have been found for this problem. In order yet to obtain an immune response against the native epitopes immunization is effected according to the invention with living bacteria or bacteria most closely approaching this condition. According to one method the bacteria are injected into the test animal together with an antibiotic in such a manner that the resulting bacteriostatic effect allows exposition of the growth

inhibited but still living bacterium to the immune system. According to a second method of obtaining an immune response to living bacteria the bacterial strain is treated with a low concentration formalin. As a probable result, the ingestion of the bacteria by phagocytes (pathogenic bacteria are often ingested by phagocytes to live on intracellularly and

invisibly to the immune system) is inhibited to such an extent that exposition to cells of the immune system may occur.

According to the invention it is therefore preferred that for the preparation of the monoclonal antibodies immunization with the living microorganisms is used in combination or after treatment with an antibiotic (in an amount inhibiting growth of the bacterium) or formaldehyde (in an amount inhibiting ingestion of the bacterium by phagocytes).

According to the invention the antibodies used for the isolation of the microorganisms to be demonstrated must be or become bound to a solid support (optionally after reaction with the microorganisms present in the sample if the

antibodies and solid support are suitably modified so as to enable realization of such a later coupling, e.g., via a biotin/avidin system, a hapten/antihapten system etc.).

Although the nature of the solid support is basically not subjected to special restrictions and the solid support may consist of, e.g., inert particles of metal, plastic or glass that can be separated from the rest of the sample by

centrifugation, it is highly preferred according to the invention to use magnetic particles that are very easy to pick up from the sample by means of a magnetic force. As compared with the use of the wall of a tube as solid support for the antibodies, the use of particles and especially magnetic particles requires, due to a proper contact with the sample, much less time to realize optimum binding of microorganism and antibody.

According to the invention it is therefore strongly preferred to use magnetic particles as solid support for the antibodies, by means of which the microorganisms to be demonstrated are isolated from the sample. Most preferably, the microorganisms to be demonstrated are isolated from the sample by adding to the sample magnetic particles to which are bound monoclonal antibodies having specific affinity for the microorganisms to be demonstrated and separating after incubation the magnetic particles with, if present, the microorganisms to be demonstrated bound thereto. It is further preferred to use magnetic particles with monoclonal antibodies bound thereto having specific affinity for the microorganisms to be demonstrated, which have been obtained by incubating the monoclonal antibodies with magnetic particles coated with antibodies having specific affinity for the monoclonal antibodies. If the monoclonal antibodies are immunoglobulins, e.g., from the mouse, the magnetic particles may be coated with, e.g., immunoglobulins of the goat having specificity for immunoglobulins of the mouse.

Bacteria and other microorganisms show the effect of aspecific binding or attachment to solid surfaces. This particularly constitutes a great problem if, in addition to the microorganisms to be demonstrated, the sample to be examined also contains other species of bacteria, such as

Salmonella. This problem is further increased if a small number of the microorganisms to be demonstrated is present besides a large excess of other microorganisms, e.g., one

Salmonella bacterium per one million of Escherichia coli bacteria. In that case the method disclosed in U.S. patent 4,677,055 will fail because the Salmonellas are completely overgrown by the Escherichia coli bacteria attaching to the particles. The present invention fully solves this problem by using PCR as method of detection. By a proper selection of the primers this ensures specificity and a high sensitivity. To obtain these desirable properties, however, it is advisable to effect PCR at the DNA released from the cells and separated from the solid support, which DNA is obtained by lysing the bacteria (e.g., by heating to 60-100°C) and using the

supernatant for PCR after centrifugation. According to the invention it is therefore preferred to isolate the DNA from the organisms bound to the magnetic particles and to subject it to PCR in the absence of the magnetic particles. For the rest, the invention demands little of the sample recovery for PCR; using lysis buffer, phenol extractions and CsCl gradients is not necessary, not even if the method is directed to demonstrating, e.g., Enterobacteriaceae or Listeria.

The PCR conditions are known per se to a skilled worker (see, e.g., U.S. patent 4,683,202), as are the species of DNA polymerase suitable therefor, such as the frequently used Taq DNA polymerase. The selection of the primers, however, is specific for the present method: by this selection it can be ensured that the method is specific for a certain genus, a certain species or even a certain strain of a microorganism. To find suitable primers, however, is not always very easy. This proves to be a problem, e.g., if specificity for a certain genus of bacteria is to be ensured, e.g., for the genus Salmonella, i.e. if it is wished that all species and strains belonging to the genus Salmonella be recognized with the method while bacteria belonging to other genera are not recognized. It is known that rRNA sequences are highly similar within a certain genus, and this has already been utilized for hybridization experiments (see EP-A-0 357 306; EP-A-0 277 237 ; WO 88/03957). The use of rRNA primers has already been

described by Chen et al, FEMS Microb. Lett. 57, 1989, 19-24, but for demonstrating bacteria in general. The use of rRNA primers is a possibility comprised by the invention, but is not suitable in all cases. For particularly in case of

Salmonella a rather strong sequence variation proves to occur in the rRNA sequences yet. A suitable alternative has been found in the sequence of the "origin of DNA replication" of the bacterial chromosome (oriC). Of a number of

Enterobacteriaceae the sequence of oriC is known (Kornberg in

"Supplement to DNA replication", Freeman and Co., San

Francisco). This oriC sequence consists of regions conserved between the Enterobacteriaceae and portions that are variable. Unknown, however, was that these variable portions are

conserved within, e.g., the genus Salmonella and may therefore serve as the basis for primers for a genus-specific PCR.

The invention therefore comprises methods wherein primers are used in PCR that are based on portions of an rRNA

sequence. This approach has been found useful in practice for, e.g., bacteria of the genus Listeria.

The invention also comprises methods wherein primers are used in PCR that are based on portions of a sequence of the origin of DNA replication.

A concrete example thereof is a method wherein bacteria of the genus Salmonella are demonstrated and the following oligonucleotide primers are used in PCR:

primer 1: 5'-TTATTAGGATCGCGCCAGGC-3'

primer 2: 5'-AAAGAATAACCGTTGTTCAC-3'.

In a concrete practical example this use of the invention will be further illustrated.

A second example is a method wherein bacteria of the genus Klebsiella are demonstrated and the following

oligonucleotide primers are used in PCR:

primer 1: 5'-CTTGTCTTGTGGATAAGTCA-3^,

primer 2: 5'-TCTTCTGTGGATAACTATGC-3' It has been established by way of experiment that with this primer combination and with Klebsiella-specific

monoclonal antibodies the method according to the invention is selective for bacteria of the genus Klebsiella, which means a positive result for Klebsiella oxvtoca and Klebsiella

pneumoniae and a negative result for Escherichia _coli,

Citrobacter freundii, Serratia marcescens, Proteus mirabilis, Enterobacter cloacae and Enterobacter aerogenes.

However, not every primer combination is useful and active in PCR. Thus it has been established that for

demonstrating Klebsiella the primer combination based on the oriC sequence:

primer 1: 5'-CTTGTCTTGTGΓGATAAGTCA-3'

primer 2 : 5'-AAGTATAACCGTTGCCTGA-3'

is inactive.

In the following example monoclonal antibodies capable of binding to living salmonella bacteria have been used.

Hybridomas producing these monoclonal antibodies were

deposited with the ECACC (European Collection of Animal Cell Cultures), UK, on November 28, 1990, under the following numbers:

MAB 55-23-B7: ECACC 90112807

MAB 70-2-2A: ECACC 90112808

MAB 71-40-A1: ECACC 90112809

Example

PCR amplification

The PCR technique was used for the amplification of a 160 bp portion of the sequence of the origin of DNA replication. For this purpose primers were used having a sequence derived from the origin of replication of Salmonella typhimurium.

These oligonucleotide primers were synthesized on a Pharmacia LKB Gene Assembler Plus Synthesizer using the phosphoramidite technology. After deprotection and cleavage from the solid support the oligonucleotides were purified by chromatography on NAP10 columns. The primers had the following sequences: primer 1: 5'-TTATTAGGATCGCGCCAGGC-3'

primer 2: 5'-AAAGAATAACCGTTGTTCAC-3'

The specificity of the set of primers was tested in a PCR at 27 Salmonella strains and 19 other Enterobacteriaceae (no salmonella) (see Table A). The PCR reaction mixture (final volume 100 μl) consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.001% (w/v) gelatin, 100 μM of each dNTP, 0.5 μM of each primer, 1.25 U Ampli-Taq polymerase (Cetus, Emeryville, CA, USA). The amplification was carried out in 25 cycles on a Perkin-Elmer Thermocycle (Perkin-Elmer

Instruments, Norwalk, CT, USA). A cycle consisted of a

denaturation step of 1 min. at 94°C, a primer annealing step of 1 min. at 50°C and an extension step of 1 sec at 72°C.

After amplification 15 ml of the sample were subjected to electrophoresis on a 1.5% agarose gel. The DNA was stained with ethidium bromide. Fragments obtained by cutting lambda DNA with PstI were used as length markers (the lambda DNA and the restriction endonuclease PstI were, like the dNTPs, from Boehringer Mannheim, Germany).

After the amplification a DNA fragment of the expected size was found not only with the Salmonella typhimurium

strains, but with all tested salmonella bacteria. On the other hand, no amplification occurred with the tested other

Enterobacteriaceae such as Shigella, Escherichia coli,

Citrobacter and Psendomonas. it was derived therefrom that the selected set of primers is Salmonella-specific.

Sensitivity and specificity of the method

A dilution series of Salmonella typhimurium, which varied from 5×10⁷ to 5×10² cells/ml, was tested (each time 100 μl) in a method according to the invention. Magnisort M particles (magnetic chromium dioxide) from DuPont (Wilmington, DE, USA) were used as magnetic beads. These were coated with

immunoglobulins of the goat which were specific for igG and IgM of the mouse. The binding of monoclonal antibodies (of subclass IgM), directed against Salmonella bacteria, to the magnetic beads occurred by incubating supernatant of the hybridoma culture with the coated magnetic beads for 5 min at 35°C in a phosphate buffered saline with 1% gelatin (gPBS). The magnetic particles were isolated by means of a magnet, after which the supernatant was thrown away.

The test samples were suspended in gPBS and added to the magnetic particles. After incubation of 15 min at 35°C the magnetic particles were isolated by means of a magnet and washed three times with gPBS. After the last washing step the magnetic particles were resuspended in aqua bidest.

In an attempt to carry out PCR directly at the bacteria extracted with the magnetic particles it turned out that the amplification was inhibited by the magnetic particles. For this reason the samples were incubated for 5 min at 100°C to destroy the bacterial cells and centrifuged for a short period of time, after which a fraction of the supernatant (with bacterial DNA therein) was subjected to PCR. PCR (35 DNA amplification cycles) was carried out in the above manner.

As a result (not shown) it was found that an amount of cells of 10³ could be detected by means of the method

according to the invention. In order to determine the

efficiency of the binding of the Salmonella bacteria to the magnetic particles coupled to the monoclonal antibodies, the supernatant obtained after incubation with the bacteria was also subjected to PCR. No amplification of the target DNA sequence was found, from which it can be derived that the binding of the Salmonella bacteria to the monoclonal

antibodies on the magnetic particles was nearly complete.

Further experiments showed that the method according to the invention was also capable of demonstrating 10³ Salmonella bacteria in a sample which also contained 10⁷ non-Salmonella bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca and Citrobacter freundii). In control experiments with Escherichia coli. Pseudomonas aeruginosa, Klebsiella oxytoca, Citrobacter freundii and gPBS no detectable amplification was observed. TABLE A PCR amplification of Salmonella strains and other Enterobacteriaceae strains

PCR Salmonella amplification sero group

S. durazzo + A

S. clinical isolate M + A

S. paratyphi A-0 + A

S. typhimurium L + B

S. typhimurium 1724 + B

S. typhimurium 14H + B

S. bredeny + B

S. derby + B

S. heidelberg + B

S. brandenburg + B

S. clinical isolate 10.2 + B

S. saintpaul + B

S. paratyphi B + B

S. abortus equi + B

S. infantis + Cl

S. newport + C2

S. belem + C

S. clinical isolate 859 + Cl

S. paratyphi C + C

S. panama + D

S. typhosa + D

S. enteriditis + D

S. shargani + E

S. give + Ξ

S. Pretoria + F

S. atlanta + G

S. worthington + G

Escherichia coli - Escherichia coli 07Kl - Klebsiella pneumonia 1.100 - Klebsiella pneumonia 1.88 - Klebsiella oxytoca - Citrobacter freundii - Citrobacter diversus - Serratia liquefaciens - Serratia marcescens - Enterobacter agglomerans - Enterobacter aerogenes - Enterobacter cloaceae - Proteus mirabilis - Morganella morganii - Proteus vulgaris - Pseudomonas aeruginosa - Shigella flexneri - Shigella dysenteriae - Yersinia enterolitica -

Claims

1. A method for demonstrating microorganisms in a sample wherein the microorganisms to be detected are isolated from the sample by means of antibodies having a specific affinity for these microorganisms and a solid support for these antibodies, and subsequently the DNA of the thus isolated microorganisms is subjected to a PCR (polymerase chain reaction) in which primers specific for the microorganisms to be demonstrated are used.

2. A method as claimed in claim l, wherein the

microorganisms to be demonstrated consist of bacteria, fungi or yeasts of a certain genus, a certain species or a certain strain.

3. A method as claimed in claim l, wherein the

microorganisms to be demonstrated consist of bacteria of one of the genera Salmonella, Klebsiella or Listeria.

4. A method as claimed in claim 1, wherein for the

isolation of the microorganisms to be demonstrated from the sample monoclonal antibodies are used which have been

generated against the living microorganism.

5. A method as claimed in claim 4, wherein for the

preparation of the monoclonal antibodies immunization with the living microorganisms is used in combination or after

treatment with an antibiotic (in an amount inhibiting growth of the bacterium) or formaldehyde (in an amount inhibiting ingestion of the bacterium by phagocytes).

6. A method as claimed in claim 1, wherein magnetic particles are used as solid support for the antibodies, by means of which the microorganisms to be demonstrated are isolated from the sample.

7. A method as claimed in claim 1, wherein the

microorganisms to be demonstrated are isolated from the sample by adding to the sample magnetic particles to which are bound monoclonal antibodies having specific affinity for the microorganisms to be demonstrated and separating after incubation the magnetic particles with, if present,

microorganisms to be demonstrated bound thereto.

8. A method as claimed in claim 7, wherein magnetic particles with monoclonal antibodies bound thereto having specific affinity for the microorganisms to be demonstrated are used, which have been obtained by incubating the

monoclonal antibodies with magnetic particles coated with antibodies having specific affinity for the monoclonal antibodies.

9. A method as claimed in claim 1, wherein the DNA is isolated from the microorganisms bound to the magnetic particles and is subjected to a PCR in the absence of the magnetic particles.

10. A method as claimed in claim 1, wherein primers are used in PCR that are based on portions of an rRNA sequence.

11. A method as claimed in claim 1, wherein primers are used in PCR that are based on portions of a sequence of the origin of DNA replication.

12. A method as claimed in claim 1, wherein bacteria of the genus Salmonella are demonstrated and the following

oligonucleotide primers are used in PCR:

primer 1: 5'-TTATTAGGATCGCGCCAGGC-3'

primer 2: 5'-AAAGAATAACCX3TTGTTCAC-3'.

13. A method as claimed in claim 1, wherein bacteria of the genus Klebsiella are demonstrated and the following

oligonucleotide primers are used in PCR:

primer 1: 5'-CTTGTCTTGTGGATAAGTCA-3'

primer 2: 5'-TCTTCTOTGGATAACTATGC-3' .

14. A kit for demonstrating microorganisms in a sample, comprising antibodies having a specific affinity for the microorganisms to be detected, which antibodies are bound to a solid support for these antibodies or are provided with an agent by means of which they can be bound to a solid support for the antibodies likewise belonging to the kit, as well as a set of primers for a PCR (polymerase chain reaction) that are specific for the microorganisms to be demonstrated.

15. A kit as claimed in claim 14, comprising monoclonal antibodies having specific affinity for the microorganisms to be demonstrated, generated against the living microorganism.

16. A kit as claimed in claim 15, comprising monoclonal antibodies prepared using an immunization with the living microorganism in combination or after treatment with an antibiotic (in an amount inhibiting growth of the bacterium) or formaldehyde (in an amount inhibiting ingestion of the bacterium by phagocytes).

17. A kit as claimed in claim 14, comprising magnetic particles as the solid support for the antibodies.

18. A kit as claimed in claim 17, comprising magnetic particles with monoclonal antibodies bound thereto having specific affinity for the microorganisms to be demonstrated, obtained by incubating the monoclonal antibodies with magnetic particles coated with antibodies having specific affinity for the monoclonal antibodies.

19. A kit as claimed in claim 14, comprising a set of primers for PCR based on portions of an rRNA sequence.

20. A kit as claimed in claim 14, comprising a set of primers for PCR based on portions of a sequence of the origin of DNA replication.

21. A kit as claimed in claim 14 for demonstrating bacteria of the genus Salmonella, comprising the following

oligonucleotide primers for PCR:

primer 1: 5'-TTATTAGGATCGCGCCAGGC-3'

primer 2: 5'-AAAGAATAACCGTTGTTCAC-3'.

22. A kit as claimed in claim 14 for demonstrating bacteria of the genus Klebsiella, comprising the following

oligonucleotide primers for PCR:

primer 1: 5'-CTTGTCTTGTGGATAAGTCA-3'

primer 2: 5'-TCTTCTGTGGATAACTATGC-3'.