KR20060116816A - Molecular detection of mycobacterium avium subsp. paratuberculosis - Google Patents

Abstract

The present invention refers to the molecular detection and identification of the Mycobacterium avium subsp. Paratuberculosis (MAP). More particularly, the invention is effected with the development of PCR assays with one or more oligonucleotide primers targeted to different genomic regions of the IS900 element specific for MAP.

Description

MOLECULAR DETECTION OF MYCOBACTERIUM AVIUM SUBSP. Mycobacterium avium subspecies paratuberculosis. PARATUBERCULOSIS}

The present invention is Mycobacterium avium subspecies paratumberculosis ( Mycobacterium avium subsp. paratuberculosis , "MAP"). More specifically, it relates to PCR analysis using oligonucleotide primer pairs that target different genomic regions of IS900 elements specific to MAP.

Mycobacterium avium subspecies paratuberculosis (MAP), which can cause serious and sometimes fatal diseases in many animal species, namely paratuberculosis, is a very difficult bacterium in terms of microbial identification. Moreover, there is an increasing number of reports linking this particular bacterial MAP to the etiology of certain forms of human infectious enteritis <Chiodini, 1989; Chamberlin et al., 2001>.

The identification of MAPs today is facilitated by the widespread use of molecular technology. However, because of the need for finding results, current technology is often difficult and practically only used for research purposes.

More specifically, by serology Diagnosis of MAP infections requires confirmation again to avoid false negative results often recorded in the early stages of infection and cross-reaction with naturally occurring and genetically related mycobacterial species (Ridge et al.), 1991; Nielsen et al., 2001>. The usefulness of culture as a diagnostic or confirmation method is reduced because of the long incubation period required for the growth of MAP.

Therefore, polymerase chain reaction ("PCR") has long been thought to be a very useful tool for the identification and detection of such bacteria (Hance et al., 1989). However, confirmation of the results is also necessary for molecular techniques, especially when used for routine diagnosis. It is usually proposed to include hybridization assays, but this makes the whole process difficult and practically only used for research purposes.

Therefore, low cost fast PCR methods that can be reliably incorporated into routine MAP identification from clinical materials are of great importance. Even at low cost, they must have very high sensitivity and specificity. It is also very important to ensure that the results can be repeated very often, especially when the methods that actually work satisfactorily in one laboratory can often produce useless results.

<Overview of invention>

The present invention has enabled the development of low cost, rapid PCR based methods that can be reliably incorporated into the routine identification of MAP from clinical or other materials.

Crosschecks were performed internally in the laboratory to ensure the authenticity of the proposed assay and the reproducibility of the results in other laboratories.

The invention has allowed the development of methods for MAP identification and routine detection from tissue samples of human, animal, plant or other origin. The method may be applied not only to clinical materials, but also to other materials of human, animal or plant origin, dust, foodstuffs, and the like.

The proposed analysis can be applied to other laboratories by combining high reproducibility independent of a variety of specific conditions and practices at optimal performance and optimal cost.

The present invention is realized through the development of PCR assays using one or more of the following oligonucleotide primers.

In a preferred embodiment, a combination of oligonucleotide primers was used to form a set of oligonucleotides. Preferred sets of oligonucleotides are P1N / P3N, P1N / P4N, P2N / P3N, P2N / P4N, P1N / P2N / P3N, P1N / P2N / P4N, P1N / P3N / P4N, P2N / P4N. More preferred is an oligonucleotide set comprising all four oligonucleotide primers, ie P1N / P2N / P3N / P4N.

The primers target genomic regions of the IS900 element specific to MAP and allow efficient, fast and reliable detection of Mycobacterium avium subspecies paratuberculosis (MAP). These primers were selected from many because they show better results.

In one-tube PCR analysis for robust detection of MAP, it has been found to be desirable to use the oligonucleotide primers alone or in combination.

In a preferred embodiment, oligonucleotide primer combinations were used to form oligonucleotide sets. Such oligonucleotide sets useful for MAP detection are P1N / P2N, P3N / P4N, P1N / P3N, P1N / P4N, P2N / P3N, P2N / P4N, P1N / P2N / P3N, P1N / P2N / P4N, P1N / P3N / P4N , P2N / P3N / P4N.

In a particularly more preferred embodiment, an oligonucleotide set comprising all four oligonucleotide primers, namely P1N / P2N / P3N / P4N, was used. More preferably, one-tube nested PCR is used with all four oligonucleotide primers, ie P1N / P2N / P3N / P4N.

In order to find a reliable method for detecting MAP using PCR, tests were conducted in a number of laboratories, some of which are located in other countries. In order to detect the reliability and accuracy of the method, usually all other parameters constituting the PCR reaction did not match and this factor was also included for the evaluation of our method. This approach has not been described previously in relation to MAP.

Other laboratories were in charge of evaluating DNA extraction procedures and other PCR assays for the detection of MAP. Starting with the evaluation of primer specificity through extensive GenBank database searches for PCR, one in-house method and one commercial method for DNA extraction, while many other assays were evaluated This was used.

Therefore, we concluded with one-tube inclusions, two-tube inclusions, and single, PCR analysis targeting different genomic regions of the IS900 element specific to MAP. The four methods were applied to positive and negative control samples, which were tissue samples collected from mycobacterium avium infected chickens and cows with paratuberculosis immobilized on formalin and stained with paraffin. formalin-fixed paraffin embedded tissue sample ("FFPE") and pure bacterial cultures. In the test performed all samples were identified by code number and the results were shared among cooperating laboratories. Based on the criteria of certainty and cost, the better performed procedure was a one-tube inclusion PCR analysis using the oligonucleotide primers in combination with in-house DNA extraction methods. The method yielded the expected results from the positive and negative control samples used to allow discrimination between genetically related mycobacterial species. The agreement of the results obtained from the other laboratories where the test was performed indicates that the assay is certain under other laboratory conditions.

Table 1 shows the prototype strains of mycobacterial species used for the evaluation of the proposed PCR method.

Table 2 shows the nucleotide sequence composition of the primers used.

Table 3 shows the temperature profiles of the B1, B2, B3 and B4 PCR reactions.

Table 4 shows the relative PCR results for FFPE samples collected from cattle with typical disorders of John's disease.

1 shows representative results by electrophoresis of PCR products of the B1, B2, B3 and B4 methods incorporated to examine FFPE samples collected from cattle and chickens.

More specifically,

1 lane represents a 100 bp <Biolabs> DNA ladder.

Lanes 2 to 4 represent DNA products of PCR performed on samples derived from the small intestine of the bovine.

Lane 5 represents the DNA product of PCR performed on samples derived from bovine mesenteric lymph nodes.

Lanes 6 to 7 represent DNA products of PCR analysis performed on samples derived from bovine ileum.

Eight lanes show PCR results generated from spleen samples of chickens.

9 lanes show PCR results generated from chicken liver samples.

T lanes represent PCR results generated from negative control samples (no DNA).

material:

DNA was extracted from the culture and clinical material (FFPE) and subjected to the molecular techniques described below.

More specifically, a culture of Mycobacterium avium subspecies Paratuberculosis (MAP) 10 strain and 30 different strains of Mycobacterium were used (as described in detail in Table 1). To assess the specificity of the described method, a number of genetically related bacterial species Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Salmonella (Salmonella) genus Enterobacter (Enterobacter) in> was used.

Table 1 below shows the number of strains of mycobacterial species used for the evaluation of the proposed PCR method.

Number of strains Bell source One M. Tuberculosis ( M. tuberculosis )  RIVM, National Institute of Public Health and Environment, The Netherlands One M. M. cannetti One M. M. bovis BCG ) 5 M. M. anium One M. M. intracellulare type I One M. M. intracellulare type II One M. M. gordonae I One M. M. fortuitum One M. Scrofulaum ( M. scrofulaceum ) One M. M. kansasii One M. Abium paratuberculosis ( M. avium paratuberculosis ) 8 M. Tuberculosis ( M. tuberculosis )  Thoracic Disease Hospital, Greece 3 M. M. avium 3 M. M. gordonae One M. M. celatum One M. M. fortuitum 9 M. Abium paratuberculosis ( M. avium paratuberculosis ) VRI Veterinary Research Institute, Czech Republic

The clinical material (tissue sample) included in the tests performed consisted of FFPE samples collected from cattle and chickens. Ten intestinal FFPE samples collected from cows with typical disorders of paratubulocysis were used. The same number of liver and intestinal FFPE samples were used from chickens with typical disorders of mycobacterial infection. Clinical material from the corresponding cows and chickens was positive for the cultures of MAP and Mycobacterium avium subspecies Avian (MAA) respectively. In addition, 30 raw FFPE intestinal and lymph node samples collected from humans and animals with no clinical or other signs of mycobacterial infection were examined. This material was used as a negative control (this data is not shown on the specification).

Way:

A) DNA extraction from FFPE tissue samples and bacterial culture

DNA extraction from FFPE samples was performed in two ways, both of which were used for 2-3 paraffin sections 10 μm thick per sample.

A1. For the extraction of DNA performed as known in-house method, Ikonomopoulos et al., 1999, the material was subjected to xylene at 60 ° C., 100% ethanol and finally 75% ethanol. The wax was removed by repeated incubation at. The product of this process is incubated in SDS and proteinase K at 50 ° C. for about an hour and then degraded.

These proteins were removed by the continuous addition of phenol and phenol-chloroform-isoamyl alcohol solution. The DNA is then precipitated by ethanol and sodium acetate (Sambrook et al., 1989), collected in the form of a precipitate and eluted in 50 μl of TE buffer.

A2. Alternatively, DNA extraction from FFPE samples was performed using a MicroLysis kit (Microzone), Microzone, according to the manufacturer's instructions.

DNA extraction from bacteria was performed using CTAB-proteinase K followed by a combination of phenol and phenol-chloroform-isoamyl alcohol. The DNA extract was precipitated by ethanol and sodium acetate as above.

Quality assessment of the DNA extract in terms of volume, purity and completeness was performed by optical density counts and agarose gel electrophoresis.

B) Polymerase Chain Reaction (PCR)

A number of PCR assays were evaluated, starting with evaluating the specificity of the primers by searching the GenBank database expanded with NCBI Blast. As a result, four different PCR analyzes using a total of seven pairs of oligonucleotide primers targeting different genomic regions of the IS900 element specific to MAP were concluded and evaluated. These other PCR assays are described below in B1, B2, B3 and B4. More specifically:

B1. First control PCR analysis used IS900 urea-F and IS900 urea-R (Table 2) oligonucleotide primers. These primers amplify the 707 bp DNA fragment of the IS900 element of MAP (Green et al., 1989).

Table 2 below shows the composition of the oligonucleotide primers.

B2. Another control PCR analysis was an inclusion PCR analysis using s204 and s749 (Table 2) primers to amplify 563 bp DNA fragments of IS900 elements of MAP. The product of this reaction diluted to 1:10 is mixed into a second reaction using s345 and s535 (Table 2) primers that amplify 210 bp DNA fragments in 563 bp DNA fragments amplified by s204-s749 primers. Englund et al., 1999>.

B3. Another control PCR analysis was an inclusion PCR analysis using IS01 and IS04 (Table 2) primers to amplify 302bp DNA fragments of IS900 elements. The product of this reaction diluted 1:10 is mixed into a second reaction using IS02 and IS03 (Table 2) primers which amplify the 159 bp DNA fragment in the previously amplified 302 bp DNA fragment.

B4. Finally, one PCR assay according to the present invention was a one-tube embedded PCR assay comprising P1N, P2N, P3N and P4N (Table 2) primers. All primers are added simultaneously into the PCR reaction mixture to amplify the 257 bp DNA fragment of the IS900 element.

The reaction mixture for each of the PCR assays was 50 mM KCl, 10 mM Tris-HCl (pH 8.4 at room temperature), 1.5 mM MgCl ₂ , oligonucleotide primers 0.25 μM, 200 μM dNTPs, Taq polymerase <Promega> 2.5 Units were prepared with 0.05-0.1 μg DNA and with an amount of HPLC quality water to bring the final volume to 50 μl for B1 and B4 assays and 25 μl for B2 and B3 assays.

The temperature profile used for the B1 to B3 analysis (Table 3) is the denaturation step of the first 5 minutes at 94 ° C., then 1 minute at 94 ° C., 1 minute at 63 ° C. (for B1 and B2 analysis) or (B3 analysis). For 35 cycles of 1 minute at 62 ° C and 1 minute at 72 ° C. After this step there was a 10 minute incubation step at 72 ° C. to complete the DNA product (Table 3). In contrast to the B4 assay, which included a different temperature profile for each of the two stages of the nested reaction (Table 3), both stages of the B2 and B3 nested reactions were performed at the same temperature profile. The first step is the initial denaturation step at 94 ° C. for 1 minute, then 1 minute at 94 ° C., 1 minute at 54 ° C., 16 cycles per minute at 72 ° C., and 1 minute at 94 ° C., 1 minute, 67 ° C. 30 cycles of 1 minute at &lt; RTI ID = 0.0 &gt; C, &lt; / RTI &gt; The PCR product was analyzed by electrophoresis on 2% agarose gel, stained with ethidium bromide, and photographed.

Table 3 below shows the temperature profiles of the B1, B2, B3 and B4 reactions.

* B1 reaction was carried out in one step.

Evaluation of the sensitivity of each of the methods was determined as known (Ikomonopoulos et al., 1998) by applying the methods to a 10-fold dilution of DNA extracted from the MAP culture.

Evaluation of Reproducibility:

The DNA extraction method and PCR analysis were applied to the reference and clinical materials, respectively, under different specific conditions <type of thermocycler, type of DNA polymerase, buffer> in different laboratories. The samples were identified using code numbers and the results were exchanged and compared with other laboratories.

<Result>

A. DNA Extraction:

The A1 and A2 methods were used, which produced sufficient DNA both qualitatively and quantitatively from FFPE samples. The amount of DNA produced by 2-3 micron sections of 10 μm thick was evaluated at 1 μg / μl and the quality was the same regardless of the method used for the DNA extraction in terms of completeness and purity. However, the in-house method A1 proved to be considerably lower cost despite the more difficulties.

The quality of the DNA product extracted from the bacterial culture using the in-house method (A1) was very satisfactory with respect to the amount and integrity of the product as expected.

B. PCR

By applying the four PCR assays (B1, B2, B3 and B4) to the DNA extracted from the used bacterial culture (Table 1), unique detection and identification of all MAP strains used was possible. Negative controls, on the other hand, did not produce positive results with any of the primer pairs evaluated with respect to both bacterial culture and FFPE samples. In ten FFPE samples from cattle with John's disease, the number of positive results recorded using the methods of B1, B2, B3 and B4 was 6, 9, 6 and 10, respectively (Table 4, FIG. 1). ).

Table 4 below shows the PCR results of the B1, B2, B3 and B4 methods on FFPE samples collected from cows with typical disorders of John's disease.

PCR analysis Explanation Positive result B1 PCR 6/10 (60%) B2 Nested PCR 9/10 (100%) B3 Nested PCR 6/10 (60%) B4 Single Tube Inclusion PCR 10/10 (100%)

The sensitivity of all the PCR methods evaluated was satisfactory, especially with regard to the inclusion assay. The minimum amount of DNA required for positive results from bacterial culture was estimated at 8 pg (Method B4) for about 1500 bacterial cells (Baess et al., 1984).

C. Evaluation of Reproducibility

The method of B1 to B4 was applied to detecting MAP from bacterial cultures and FFPE tissue samples to obtain perfectly consistent results in all participating laboratories.

More specifically, it has been found that the methods of B2 and B3 can also be applied to the entire area of MAP with regard to diagnosis, only in combination. The same problem was found in the method of B1. The method of B4 enabled the detection of MAP regardless of the material used.

According to our experimental data, the method of B4, single-tube embedded PCR allows for the direct checking of results with minimal time and minimal cost, while at the same time having the highest sensitivity, specificity and reproducibility. The methods of B1 and B4 have been found to specifically detect MAP in bacteria extracted from culture. However, despite the fact that all primers were designed to detect MAP and their synthesis was based on the same genomic region (IS900 element), only the methods of B2 and B4 detected all positive FFPE samples used. This fact is another important aspect of the present invention, which demonstrates the justification of the designed evaluation procedure, while allowing the method of using the primers according to the present invention to ensure minimal false results not only for research but also for reliable routine applications. Implies a parameter.

The error of the results recorded by the methods of B1 and B3, which has been demonstrated by laboratory internal collaboration, is not surprising. The method of B3 was specifically designed to realize and increase the diagnostic efficiency of the B2 method, whereas the method of B1 was thought to be less sensitive than the above nested responses. This proved to be actually correct because the combination of the methods of B2 and B3 did not exactly identify all our positive control FFPE samples (FIG. 1).

Errors in the results recorded by the methods of B1 and B3 may also be based on the genetic diversity of the MAP strain, at least with respect to the FFPE samples evaluated. Since the same DNA product was used for all PCR reactions (also in the methods of B2 and B4), the presence of PCR inhibitors that can often produce false negative results, especially in FFPE samples. Hermon-Taylor et al. , 2000> cannot be the basis of the error. For the same reason, the results cannot be based on the possibility of DNA fragmentation. The product of the DNA extraction procedure is always assessed by electrophoresis and spectrophotometry, and samples producing poor quality DNA are always discarded. Finally, the selection of the IS900 element as a common target for all of the above PCR reactions should be excluded from factors that may lead to inefficiencies of the methods of B1 and B3. Since the demonstration of the existence of IS900 elements is considered essential for the molecular identification of MAP today (Green et al., 1998), the selection of such specific DNA target regions is essential.

Regardless of the fact that both methods of B2 and B4 are known to be sufficient for diagnosis, the method of B4 represents an important advantage over the method of B2. More specifically, the method of B2 is a PCR analysis consisting of two successive steps. In order to proceed from one stage to another, the tubes used in the first stage must be opened, which is found in large numbers in the laboratory, where the DNA product from the positive assay of the previous stage is likely to enter the mixture of the final reaction. Therefore, it is dangerous due to the infection <carry-over effect>. In contrast, this fact, which often causes an increase in the false positive result, can be avoided by the method of B4, where all components of the inclusion assay are injected together into the reaction tube and the tube is not opened during the analysis. In addition to the above, the method of B4 has important advantages over the method of B3 as well as the method of B3, which requires less consumables when the method of B4 contains one reaction, resulting in less time and more This is because it requires less cost.

The above advantages of the method of B4, and the fact that the results are controlled without the need for internal hybrids that significantly increase the cost and complexity of the assay, as well as the other PCR assays mentioned above, as well as DNA hybridization, which in particular increases the time required for the procedure, are evaluated. Make up the method of B4 which is definitely superior to all other methods that have been done.

In addition to the above, the method of B4 showed a very high sensitivity, which can very low the percentage of false negative results. Moreover, the fact that the proposed analysis (B4) only detected MAP from all the materials used, also shows very high specificity which can very low the percentage of false negative results. In this regard, it is important that the assay showed clear specificity not only for bacteria extracted from the culture but also for bacteria extracted from chicken-origin FFPE samples infected with Mycobacterium avium, which has a particularly high genetic association with MAP. Do. Contrary to the inefficiency expected in some of the other methods evaluated, the complete reproducibility of the results recorded by laboratories collaborating with the method of B4 increases certainty and substantiates the contents.

In conclusion, it can be appreciated that the nested PCR assay B4 is characterized by speed and relatively low cost and makes it possible to detect MAP from FFPE samples. The latter was demonstrated not only for our bacterial culture but also for MAA-positive FFPE samples from chicken, despite the high genetic similarity with MAA and MAP. Eventually, the B4 response is the most promising method for reliable routine diagnosis of MAP due to the agreement of the results obtained by the cooperative laboratories using the method (B4).

Given the technical basis of the PCR reaction, if the DNA extraction procedure produces a query DNA similar to the above, the proposed method using the specific nucleotide primers may be of the same value (sensitivity-specificity) for samples other than clinical material. Can produce results. Optimal performance of the proposed assay using this specific oligonucleotide primer was demonstrated by detecting MAP from cheese and milk samples collected in Greece and the Czech Republic and evaluated using other PCR assays and cultures.

<References>

-Baess I., Determination and re-examination of genome sizes and base ratios on deoxyribonucleic acid from mycobacteria, Scta Pathol Microbiol Immunol Scand Sect 1984, B92: 209-211.

Chamberlain W., Graham DY, Hulton K., El-Zimaity HM, Schwartz Naser S. .), Shafran I., El-Zaatari FA., Mycobacterium avium subsp. paratuberculosis one cause of Crohn's disease, Aliment Pharmacol Ther 2001, 15: 337-346.

-Chiodini RJ., Crohn's disease and the mycobacterioses: a review and comparison of two disease entities, Clin Micro biol Rev, 1989 Jan: 2 (1): 90-117, Review.

Englund S., Balagi-Pordany A., Bolske G., Johansson KE., Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis , Diagn Microbiol Infect Dis 33, 163-171.

Gazouli M., Ikonomopoulos JA., Zacharatos P. et al., Incidence of Mycobacterium. avium subsp. paratuberculosis in soft cheese in Greece, Proceedings of the 8th international conference on environmental science and technology, Lemnos, Greece, 2003 A pp 240-244.

Hans AJ, Grandchamp B., Levy-Frebault V., Lecossier D., and Lausier J. Rauzier J.), Bocart D., Giquel B., Detection and identification of mycobacteria by amplification of mycobacterial DNA, Mol. Microbiol 1989, 3: 843-849.

Hermon-Taylor J., Bull TJ., Sheridan J., Cheng J., Stellakis ML., Sumamar Sumar N., Causation of Crohn's disease by Mycobacterium avium subsp. paratuberculosis , Can J Gastrenterol 2000, 14: 521-539.

Ikonomopoulos JA., Gorgoulis VG., Zachartos PV. Et al., Multiplex PCR assay for the detection of mycobacterial DNA directly from sputum, In Vivo 1998; 12: 547-552.

Ikonomopoulos JA., Gorgoulis VG., Zachartos PV. Et al., Multiplex polymerase chain reaction for the detection of mycobacterial DNA in cases of tuberculosis and sarcoidosis, Mod Pathol 1999; 12: 854-862.

-Ikonomopoulos JA., Kokotas S., Gazouli M., Zavras A., Stoziou M. Stoitsiou M.), Gorgoulis V., Molecular diagnosis of leishmaniosis in dogs, Comparative application of the traditional diagnostic methods and the proposed assay on clinical samples, Veterinary Parasitol 2003; 113/2: 99-113.

Green EP., Tizard MLV., Moss MT., Thompson J., Winterbourne DL. McFadden JJ., Hermon-Taylor J., Sequence and characteristics of IS900 element, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis , Nucl. Ac Res 17, 9063-9073.

Nielsen SS., Houe H., Thamsborg SM., Bitsch V., Comparison of two enzyme-linked immunosorbent assays for serologic diagnosis of paratuberculosis (Johne's disease) in cattle using different subspecies strains of Mycobacterium avium , J Vet Diagn Invest. 2001 Mar; 13 (2): 164-6.

-Ridge SE., Morgan IR., Sockett DC., Collins MT., Condron RJ., Skillbeck End You. (Skilbeck NW.), Webber JJ., Comparison of the Johne's absorbed EIA and the complement-fixation test for the diagnosis of Johne's disease in cattle, Aust Vet J. 1991 Aug: 68 (8): 253-7, Erratum in: Aust Vet J 1991 Dec; 68 (12): 399.

Sambrook J., Fritsche EF., Maniatis T., Molecular Cloning, A laboratory Manual, Cold Spring Harbor Press, 1989, 2nd Edition.

<110> IKONOMOPOULOS, Joannis <120> Molecular Detection of Mycobacterium Avium subsp. Paratuberculosis <130> pt / 03/421 <150> GR 20030100421 <151> 2003-10-16 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 1 cgaatgaccg atggtttgag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 2 ctaagagtta ctgcaacagt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 3 gggtgtggcg ttttccttcg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide primer <400> 4 tcctgggcgc tgagttcctc 20

Claims (10)

An oligonucleotide, which is useful for the detection of Mycobacterium avium subspecies name Paratuberculosis (MAP) and is selected from the group consisting of oligonucleotides of the following sequence.

A set of oligonucleotides comprising one or more combinations or only one of the oligonucleotides according to claim 1, said combination comprising P1N / P2N, P3N / P4N, P1N / P3N, P1N / P4N, P2N / P3N, P2N / P4N, P1N / P2N / P3N, P1N / P2N / P4N, P1N / P3N / P4N, P2N / P3N / P4N and P1N / P2N / P3N / P4N. A method of detecting one or more selected from the group consisting of MA and MAP in a sample, (a) isolating DNA from the sample; (b) performing one-tube PCR from one or more of the oligonucleotides of claim 1 using the isolated DNA; (c) screening for positive PCR amplification results; (d) identifying a sample comprising MAP; The method of claim 1, wherein at least one of the oligonucleotides according to claim 1 is used for PCR. Method according to claim 3, characterized in that one or more sets of oligonucleotides according to claim 2 are used, in particular one set comprising all four oligonucleotides. Use of the method according to claim 3 or 4, wherein the animal, including a sample from a living animal, a human or an organism comprising an animal, is an inanimate organism comprising a product, dust derived from an organism including cattle, poultry or chickens. And MAP detection in at least one selected from the group consisting of samples derived from plants. Use of the method according to claim 3 or 4, for the diagnosis of MAP infection in a organism comprising a human and comprising a cow, poultry or chicken. A kit for detecting MAP in a subject, (a) means for separating DNA from the sample; (b) a set of oligonucleotides according to claim 1, a set of oligonucleotides according to claim 2, or a set of oligonucleotides according to claim 1 and an oligonucleotide of claim 2; (c) means for screening positive PCR amplification results; (d) means for identifying a sample comprising MAP Kit comprising a. A kit comprising at least one selected from the group consisting of at least one oligonucleotide according to claim 1 and at least one set of oligonucleotides according to claim 2, and a container. A kit comprising all four oligonucleotides and containers according to claim 1. Use of a kit according to any one of claims 7 to 9, for use in a method according to claim 3, 4 or 3 and 4.

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