CN102409102A - PCR primer and method for identifying mycobacterium bovis - Google Patents

Abstract

The present invention provides 5 pairs of primers for PCR identification of Mycobacterium bovis and an identification method. The 5 pairs of primers are respectively used for amplification of the 16SrRNA conservative region of Mycobacterium bacteria, the Rv0577 gene of the Mycobacterium tuberculosis complex (MTBC), the Rv1970 of the RD7 region of Mycobacterium tuberculosis, the pncA gene of Mycobacterium bovis and the gene of the RD1 region to generate specific amplified fragments, and the nucleotide sequences are shown in SEQ ID No.1-5. The identification method of the present invention is to use the total DNA of the sample as a template, use the above 5 pairs of primers to perform PCR amplification respectively, and determine the result according to the size of the amplified band. The primers of the present invention can specifically identify Mycobacterium, MTBC, Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium bovis BCG, the detection method has good sensitivity, is simple, and is easy to operate, and can realize rapid and high-throughput detection of Mycobacterium bovis.

Description

A kind of PCR primer and method for distinguishing Mycobacterium bovis

technical field

The invention relates to biological detection and identification technology, in particular to a primer for PCR identification of mycobacterium bovis and a PCR identification method using the primer.

Background technique

Bovine tuberculosis is mainly a chronic wasting infectious disease caused by Mycobacterium bovis, which can be transmitted to humans or other animals through sick animals or their products. Most cattle infected with Mycobacterium bovis are not overtly infected or have clinical symptoms only in the late stage, so early clinical diagnosis is not easy to obtain accurate results. Pathological examination is one of the most reliable methods. Pathological examination can be confirmed based on characteristic lesions, but it must be carried out after the animal is dead or slaughtered, and the required diagnosis period is long, which takes 3-8 weeks, so it is often not used in actual work . Since milk, beef and other foods are closely related to human life, Mycobacterium tuberculosis can invade all organs of the human body and animals, and tuberculosis is the most common. Therefore, timely identification of pathogenic bacteria and reasonable prevention and control of bovine tuberculosis are conducive to ensuring public health safety.

The Mycobacterium tuberculosis complex (MTBC) includes M. tuberculosis, M. africanum types I and II, M. bovis, BCG, M. microti, M. caprae, and M. canettii. At present, the most widely used method for clinically identifying Mycobacterium bovis is allergy diagnosis, but this method has a strong non-specific reaction, that is, in a considerable number of tuberculosis allergy-positive cattle, neither characteristic lesions can be found, nor can it be detected. Mycobacterium tuberculosis is isolated, or only atypical mycobacteria are isolated, and the detection rate of pregnant cows is still low, and the positive rate decreases during repeated quarantine, so it is not very ideal for diagnosing bovine tuberculosis in actual work.

In recent years, molecular biology techniques represented by polymerase chain reaction (PCR) and real-time fluorescent PCR have developed rapidly in the rapid detection and identification of tuberculosis or paratuberculosis pathogens, but they are all aimed at Mycobacterium tuberculosis complex, tuberculosis classification Mycobacterium and Mycobacterium bovis separate strains of the method, there are also directly through the genotyping method to directly identify the various types of mycobacteria, but some of these methods only provide primers for the identification of individual strains, and some provide A lot of information, but no sensitivity test, which is not perfect for clinical application, some are not economical, and the cost is high, such as fluorescent quantitative PCR. For example, in 2002, Richard C. Huard et al. established a PCR method for identifying strains in the mycobacterium complex, which can specifically identify various bacteria in MTBC; in 2007, Zhang Lu et al. based on the specificity of Mycobacterium bovis MPB70 The gene sequence was amplified by PCR to detect MTBC in milk, with a positive rate of 40.9% and a sensitivity of 40ng/L; in 2007, Meng Xianghong and others established a multiplex PCR technology to quickly identify Mycobacterium tuberculosis and Mycobacterium non-tuberculosis. The multiplex PCR method has a high consistency with the results of BACTEC culture, which provides an effective means for the differential diagnosis of clinical tuberculosis and mycobacterial disease; A TaqMan fluorescent quantitative PCR detection method for bovine tuberculosis was developed. The above-mentioned methods only separately provide PCR-related methods for identifying a certain type of bacteria, and some use methods with high economic costs such as probe hybridization and fluorescence quantification. A report on the identification of major bacteria of the genus Mycobacteria using a complete set of primers.

Contents of the invention

The purpose of the present invention is to provide a PCR primer and a PCR identification method for identifying mycobacterium bovis.

The invention is a combination of specific primers for identifying mycobacterium bovis by PCR, and is used for identifying mycobacterium, mycobacterium tuberculosis complex, tubercle bacillus, mycobacterium bovis and mycobacterium bovis BCG strain. It can respectively amplify the 16S rRNA conserved region of mycobacterium, the Rv0577 gene of the Mycobacterium tuberculosis complex (MTBC), the Rv1970 of the RD7 region of Mycobacterium tuberculosis, the pncA gene and the RD1 region gene of Mycobacterium bovis, and generate specificity Amplified fragments.

The combination of primers for PCR identification of Mycobacterium bovis provided by the invention comprises:

1 16SrRNA-F 5'-ACGGTGGGTACTAGGTGTGGGTTTC-3';

16SrRNA-R 5'-TCTGCGATTAGCGACTAAGACTTCA-3';

2MTBC-F 5'-ATGCCCAAGAGAAGCGAATACAGGCAA-3';

MTBC-R 5'-CTATTGCTGCGGTGCGGGCTTCAA-3';

3MT-F 5'-GCGCAGCTGCCGGATGTCAAC-3';

MT-R 5'-CGCCGGCAGCCTCACGAAATG-3';

4PncA-F 5'-CTCAGCTGGTCATGTTCCCGAT-3';

PncA-R 5'-CGGTGTGCCGGAGAAGCCG-3';

5RD1-F 5'-AAGCGGTTGCCGCCGACCGACC-3';

RD1-R 5'-GAGGCGATCTGGCGGTTTGGGG-3'.

The invention provides a method for identifying mycobacterium bovis by PCR. In the method, the total DNA of a sample is used as a template, and the above primer combinations are used for PCR amplification respectively, and the result is judged by agarose gel electrophoresis after the reaction is completed.

The present invention provides a PCR method for identifying Mycobacterium bovis, further comprising: when the bacteria to be tested are unknown bacteria, using the total DNA of the sample as a template, using the first to fifth pairs of primers described in claim 2 to carry out respectively PCR amplification; or

When the bacteria to be tested are bacteria of the genus Mycobacterium, use the total DNA of the sample as a template, and use the above-mentioned 2nd to 5th pair of primers to perform PCR amplification respectively; or

When the bacteria to be tested are Mycobacterium tuberculosis complex bacteria, use the total DNA of the sample as a template, and use the above-mentioned 3rd to 5th pair of primers to perform PCR amplification respectively; or

When the bacterium to be tested is Mycobacterium tuberculosis, use the total DNA of the sample as a template, and use the 4th to 5th pair of primers above to perform PCR amplification respectively; or

When the bacteria to be tested are Mycobacterium bovis bacteria, the total DNA of the sample is used as a template, and the above-mentioned fifth pair of primers is used to perform PCR amplification; after the reaction is completed, the result is judged by agarose gel electrophoresis.

The reaction program of PCR containing 16SrRNA, MTBC, PncA primer pair is: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 40s, annealing at 60°C for 40s, extension at 72°C for 40s, 31 cycles, and finally extension at 72°C for 10 minutes; or

The reaction program of PCR containing MT primer pair is: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 40s, annealing at 61°C for 40s, extension at 72°C for 40s, 31 cycles, and finally extension at 72°C for 10 minutes; or

The reaction program of PCR containing the RD1 primer pair was: pre-denaturation at 94°C for 5 min, followed by denaturation at 94°C for 40 s, annealing at 62°C for 40 s, extension at 72°C for 40 s, 31 cycles, and finally extension at 72°C for 10 min.

The above PCR amplification reaction system, wherein the specific configuration of 20 μl reaction solution is:

The primers of the present invention and related reagents can be assembled into kits for convenient use. The kits of the invention may be formed from a plurality of compartments to accommodate and secure one or more containers such as tubes or vials. One or more of these containers may contain the primers of the present invention, either in lyophilized form or dissolved in a buffer, as required. In addition, the kit of the present invention may also include one or more enzymes/reagents for PCR reactions, as well as other components and utensils required for implementing the present invention.

The sources of samples identified using the primers and methods of the present invention include isolated strain resources or animal tissues, but are not limited thereto.

The invention provides the use of the above five pairs of primers in preparing reagents for detecting and identifying Mycobacterium bovis.

Application of PCR identification method of the present invention can identify the attenuated Mycobacterium bovis, such as BCG, and the attenuated Mycobacterium bovis is usually used as the seed source for preparing the vaccine for preventing tuberculosis, so the present invention also provides above-mentioned 5 kinds The application of the primer or the PCR method of the present invention for identifying mycobacteria in the preparation of tuberculosis prevention vaccines.

The 5 pairs of primers provided by the present invention are obtained through screening and optimization, which can gradually narrow down the scope of mycobacteria to be detected and obtain accurate related detection results. The scope of detection of mycobacteria is related to the complexity of mycobacterium genus and population classification. The relationship diagram of Mycobacterium, Mycobacterium tuberculosis complex MTBC, Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis BCG strains is shown in Figure 17.

By using the 5 pairs of primers of the present invention, respectively carry out PCR amplification on the DNA of the tested strains to generate specific amplified fragments, so that the mycobacteria can be easily and accurately identified. Moreover, the primers of the present invention only specifically amplify the target fragment, and will not amplify other regions. Moreover, this study established a detection method for Mycobacterium bovis, which can identify clinically unknown samples from the genus Mycobacterium, and gradually Identify the Mycobacterium tuberculosis complex, Mycobacterium tuberculosis, Mycobacterium bovis and the attenuated strain BCG strain obtained by passage of Mycobacterium bovis, forming a complete set of PCR methods for identifying Mycobacterium bovis. The method is simple and easy to operate, and can be completed by a PCR machine, realizing rapid and large-throughput diagnosis and detection. It is more reliable than the method of using a pair of primers to identify a single strain, and reduces the probability of false negative or false positive results during identification. It can also monitor the strains stored in the laboratory to ensure that the strains are not contaminated by other impurities during storage. bacterial contamination. The research shows that this set of PCR method system established in this study has a specificity of 100% and a sensitivity of pg level.

Description of drawings

Figure 1 shows the results of 16S rRNA primer PCR products detected by 1% agarose gel electrophoresis, the product band size is 543bp; M: molecular weight standard is 2K plus DNA marker, 1: Mycobacterium tuberculosis reference strain C68503; 2: Bovine branch Bacillus reference strain C68001; 3: BCG; 4: Mycobacterium smegmatis standard strain cm ² 155; 5: Mycobacterium avium reference strain C68201; 6: Staphylococcus (CAU0411); 7: Escherichia coli (CAU0439); 8: Blank control;

Figure 2 is the result of 1% agarose gel electrophoresis detection of MTBC primer PCR products, the product band size is 786bp, M: molecular weight standard is 2K plus DNA marker, 1: Mycobacterium tuberculosis reference strain C68503; 2: Mycobacterium bovis Reference strain C68001; 3: BCG; 4: Mycobacterium smegmatis; 5: Mycobacterium avium reference strain C68201; 6: blank control;

Figure 3 shows the results of MT primer PCR products detected by 1% agarose gel electrophoresis. The product band sizes are 1116bp, M: molecular weight standard is 2K plus DNA marker, 1: Mycobacterium tuberculosis reference strain C68503; 2: Bovine branch Bacillus reference strain C68001; 3: BCG; 4: blank control;

Figure 4 is the result of 1% agarose gel electrophoresis detection of pncA primer PCR products, the product band size is 294bp, M: molecular weight standard is 2K plus DNA marker, 1: Mycobacterium bovis reference strain C68001; 2: BCG; 3: Mycobacterium tuberculosis reference strain C68503; 4: blank control;

Figure 5 shows the results of 1% agarose gel electrophoresis detection of RD1 primer PCR products, the product band size is 200bp, M: molecular weight standard is 2K plus DNA marker, 1: BCG; 2: Mycobacterium bovis reference strain C68001; 3 : blank control;

Figure 6 shows the sensitivity detection of 16S rRNA primers 1: 4ng, 2: 400pg, 3: 40pg, 4: 4pg, 5: negative control, M: the molecular weight standard is 2K plus DNAmarker;

Figure 7 MTBC primer sensitivity test 1: 4ng, 2: 400pg, 3: 40pg, 4: 4pg, 5: 400fg, 6: Negative control M: Molecular weight standard is 2K plus DNA marker;

Figure 8 MT Primer Sensitivity Test 1: 12ng, 2: 1.2ng, 3: 240pg, 4: 48pg, 5: 9.6pg, 6: 1.92pg, 7: Negative control M: Molecular weight standard is 2K plus DNAmarker;

Figure 9 pncA primer sensitivity test 1: 4ng, 2: 400pg, 3: 40pg, 4: 4pg, 5: 400fg, 6: Negative control M: Molecular weight standard is 2K plus DNA marker;

Figure 10 RD1 primer sensitivity test 1: 25ng, 2: 4.7ng, 3: 470pg, 4: 47pg, 5: 4.7pg, 6: 470fg, 7: Negative control M: Molecular weight standard is 2K plus DNAmarker;

Figure 1116S rRNA primer identification results 1-7: the preserved strains numbered 1, 2, 3, 4, 5, 6, 7 respectively; M: the molecular weight standard is 2K plus DNA marker;

Figure 12 MTBC primer identification results 1-7: the preserved strains numbered 1, 2, 3, 4, 5, 6, and 7 respectively; M: the molecular weight standard is 2K plus DNA marker;

Figure 13 MT primer identification results 1-7: the preserved strains numbered 1, 2, 3, 4, 5, 6, and 7 respectively; M: the molecular weight standard is 2K plus DNA marker; +: positive control; -: negative control;

Fig. 14 Identification results of pncA primers 1-7: the preserved strains numbered 1, 2, 3, 4, 5, 6, and 7 respectively; M: the molecular weight standard is 2K plus DNA marker; +: positive control; -: negative control;

Figure 15 RD1 primer identification results 1-5: the preserved strains numbered 1, 2, 4, 6, and 7 respectively; M: the molecular weight standard is 2K plus DNA marker; +: positive control; -: negative control.

Figure 16 Clinically identify cat skin nodule tissue fluid PCR identification of suspected Mycobacterium tuberculosis infection;

M: Molecular weight standard is 2K plus DNA marker, 1: PCR result of 16SrRNA primer; 2: PCR result of MTBC primer; 3: PCR result of MT primer; 4: PCR result of PncA primer; 5: PCR result of RD1 primer;

Figure 17 Schematic diagram of the relationship between Mycobacterium, Mycobacterium tuberculosis complex MTBC, Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis BCG strains that can be detected by this PCR identification method

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

The bacterial strains used in the present invention are: Mycobacterium bovis reference strain C68001, Mycobacterium tuberculosis reference strain C68503, Mycobacterium bovis BCG reference strain C68017, Mycobacterium avium reference strain C68201 purchased from China Veterinary Drug Control Institute; Bacillus standard strain cm ² 155 and non-tuberculous mycobacterium clinical isolate C68203 are preserved by this experiment; staphylococcus and Escherichia coli are preserved by this laboratory, numbered CAU0411, CAU0439).

The design of embodiment 1PCR specific primer

The 16S rDNA gene corresponding to 16SrRNA is highly conserved in the bacterial genome, and can identify the principle of bacteria of the genus. Firstly, primers are designed according to the conserved region of the 16Sr RNA of Mycobacterium. The target fragment size is 543bp sequence, which is designed using software and Primer 3 Primer, the primer sequence is:

16SrRNA-F 5'-ACGGTGGGTACTAGGTGTGGGTTTC-3';

16SrRNA-R 5'-TCTGCGATTAGCGACTAAGACTTCA-3';

Rv0577 is a restricted gene of MTBC, primers were designed according to this gene, and the size of the target fragment amplified by PCR was 786bp. The primer sequences are:

MTBC-F 5'-ATGCCCAAGAGAAGCGAATACAGGCAA-3';

MTBC-R 5'-CTATTGCTGCGGTGCGGGCTTCAA-3';

Since deletion region 7 (deleted region7, RD7) does not exist in other MTBC strains, only Mycobacterium tuberculosis has this gene region, and primers are designed according to Rv1970 (lprM) in this region, and the target fragment size of PCR amplification is 1116bp. The primer sequences are:

MT-F 5'-GCGCAGCTGCCGGATGTCAAC-3';

MT-R 5'-CGCCGGCAGCCTCACGAAATG-3';

Experiments have proved that human Mycobacterium tuberculosis and Mycobacterium bovis have a SNP phenomenon on the pncA gene encoding pyriquinamide. Using the characteristics of gene point mutations, primers are designed to specifically identify Mycobacterium bovis (including BCG strains) , Mycobacterium bovis (and BCG strain) can specifically amplify a 298bp band, while Mycobacterium tuberculosis has no band. . The primer sequences are:

PncA-F 5'-CTCAGCTGGTCATGTTCCCGAT-3';

PncA-R 5'-CGGTGTGCCGGAGAAGCCG-3';

NCBI gene analysis results show that the RD1 region only exists in pathogenic mycobacteria, and the RD1 region gene is 9.5kb in length, with a total of 9 open reading frames (Rv3871-3879), so we can use this to distinguish the bovine branch Bacillus and BCG, the BCG strain lacks the RD1 region of the gene. According to the design of primers based on this gene, for Mycobacterium bovis, the RD1 sequence with a full length of 9.5kb cannot be effectively amplified, while BCG can only amplify the 200bp sequence products, so the two can be specifically identified. The primer sequences are:

RD1-F 5'-AAGCGGTTGCCGCCGACCGACC-3';

RD1-R 5'-GAGGCGATCTGGCGGTTTGGGG-3'.

The establishment of embodiment 2PCR amplification method

1. The PCR reaction system uses the total bacterial DNA as a template for PCR reaction. The 20 μL reaction system is:

2. PCR reaction conditions

The reaction program of PCR containing 16SrRNA, MTBC, PncA primer pair is: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 40s, annealing at 60°C for 40s, extension at 72°C for 40s, 31 cycles, and finally extension at 72°C for 10 minutes;

The reaction program of PCR containing MT primer pair is: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 40s, annealing at 61°C for 40s, extension at 72°C for 40s, 31 cycles, and finally extension at 72°C for 10 minutes;

The reaction program of PCR containing the RD1 primer pair was: pre-denaturation at 94°C for 5 min, followed by denaturation at 94°C for 40 s, annealing at 62°C for 40 s, extension at 72°C for 40 s, 31 cycles, and finally extension at 72°C for 10 min.

After the reaction, the amplified products were judged by agarose gel electrophoresis.

The specificity test of embodiment 3PCR method

The DNA of Mycobacterium tuberculosis C68503, Mycobacterium bovis C68001, Mycobacterium bovis BCGC68017, Mycobacterium smegmatis cm ² 155, Mycobacterium avium C68201, Staphylococcus CAU0411, and Escherichia coli CAU0439 were used as templates, and water was used as the template. For the blank control, 5 pairs of primers of the present invention were used respectively, and the PCR method established by the present invention was used for inspection, and the specific amplification band determination result of agarose gel electrophoresis after the PCR reaction was completed.

test results:

The PCR results of 16SrRNA primers: the PCR amplification products of Mycobacterium were positively amplified, while the other strains and the blank control had no target amplified fragments. The results are shown in Figure 1.

PCR results of MTBC primers: Mycobacterium tuberculosis, Mycobacterium bovis and BCG were positively amplified, while Mycobacterium smegmatis, Mycobacterium avium and the blank control had no target amplified fragments. The results are shown in Figure 2.

The PCR results of MT primers: Mycobacterium tuberculosis was positively amplified, while Mycobacterium bovis, BCG and the blank control had no target amplified fragments. The results are shown in Figure 3.

PCR results of pncA primers: Mycobacterium bovis and BCG were positively amplified, while Mycobacterium tuberculosis and the blank control had no target amplified fragments. The results are shown in Figure 4.

The PCR result of RD1 primer: BCG showed positive amplification, and Mycobacterium bovis and the blank control had no target amplified fragments. The results are shown in Figure 5.

The reaction product was subjected to 1% agarose gel electrophoresis. After the gel was recovered, it was ligated with the pGEM-T easy vector and transformed into DH5α competent cells. The recombinant plasmid was then purified by OMEGA plasmid extraction kit and sent to the sequencing company for sequencing. The results show that the 5 pairs of primers designed by the present invention have good specificity, and can specifically identify Mycobacterium, Mycobacterium tuberculosis complex, Mycobacterium tuberculosis, Mycobacterium bovis and BCG.

The sensitivity test of embodiment 4PCR method

Using Mycobacterium bovis reference strain, human Mycobacterium tuberculosis reference strain, and Mycobacterium bovis BCG reference strain as templates, the DNA concentrations were respectively detected as 4ng/μl, 12ng/μl and 47ng/μl, and serially diluted, and finally Each dilution of Mycobacterium bovis reference strain contains 400fg, 4pg, 40pg, 400pg, 4ng of DNA; each dilution of human Mycobacterium tuberculosis reference strain contains 1.2pg, 9.6pg, 48pg, 240pg, 1.2ng, 12ng of DNA DNA; each dilution of Mycobacterium bovis BCG reference strain contains 470fg, 4.7pg, 47pg, 470pg, 4.7ng, 25ng of DNA; amplify according to the above PCR system and conditions respectively, and test its sensitivity. The PCR products were electrophoresed on 1% agarose gel (see Figures 6, 7, 8, 9, 10). Result shows, 16S rRNA primer, MTBC primer, pncA primer sensitivity can detect the DNA of 40pg; MT primer sensitivity can detect the DNA of 9.6pg; ET primer sensitivity can detect the DNA of 470fg, illustrate that PCR method of the present invention has very good sensitivity.

Application of embodiment 5 PCR method

Using the established PCR method, 7 strains of mycobacteria kept in the laboratory were identified. The 7 strains of mycobacteria are:

1: C68021, Mycobacterium bovis, from the UK Central Veterinary Laboratory, AN5;

2: C68004, Mycobacterium bovis, from Harbin Veterinary Research Institute, Beijing Department;

3: C68501, Mycobacterium tuberculosis, preserved in the National Animal Spongiform Encephalopathy Laboratory of China Agricultural University;

4: C68006, Mycobacterium bovis, isolated from tuberculosis cattle, Beijing, 1953, preserved in the National Animal Spongiform Encephalopathy Laboratory of China Agricultural University;

5: C68203, Mycobacterium avium, isolated from tuberculosis chickens, kept in the National Animal Spongiform Encephalopathy Laboratory of China Agricultural University;

6: C68017, BCG from the Ministry of Health;

7:3003-13, from BCG, Chinese Center for Disease Control and Prevention.

First, 7 strains of mycobacteria were enriched and cultured, bacterial DNA was extracted, and the DNA was used as a template to amplify the 7 strains of mycobacteria using the above-mentioned 5 pairs of primers and the PCR system and conditions respectively. Electrophoresis was performed on an agarose gel (see Figures 11, 12, 13, 14, 15). The results are shown in Table 1.

Table 1 Identification results of laboratory-preserved strains

The results show that: the 7 known strains preserved in this experiment are detected by the PCR identification method established by the present invention, including 1 strain of environmental mycobacterium, 1 strain of tuberculosis mycobacterium, 3 strains of mycobacterium bovis, and 2 strains of BCG. The results are consistent with expectations, indicating that the PCR identification method established in the present invention can gradually identify mycobacteria, and the results are reliable.

The clinical application of embodiment 6 PCR method of the present invention

The Animal Hospital of China Agricultural University received a cat with suspected Mycobacterium tuberculosis infection. Because the previous owner fed the cat raw beef, it was suspected to be Mycobacterium bovis infection. The PCR method of the present invention is used for identification. Specifically, the tissue fluid was extracted from the skin nodules of the cat, the DNA of the sample was directly extracted, and the PCR method established by the present invention was used for identification. The results are shown in FIG. 16 . The identification results are shown in Table 2:

Table 2 The inventive method detects cat skin sample result

According to the identification results, the cat was infected with Mycobacterium bovis.

Claims (10)

1. one kind is used for the Auele Specific Primer combination that PCR differentiates Mycobacterium bovis; It is characterized in that; It respectively can be to the 16SrRNA conserved regions of mycobacterium species, the Rv0577 gene of mycobacterium tuberculosis complex MTBC; The Rv1970 in mycobacterium tuberculosis RD7 zone, Mycobacterium bovis pncA gene and RD1 district gene increase, and generate the specific amplification fragment.

2. PCR as claimed in claim 1 differentiates and uses combination of primers, comprising:

116SrRNA-F 5’-ACGGTGGGTACTAGGTGTGGGT?T?TC-3’；

16SrRNA-R 5’-TCTGCGATTAGCGACTAAGACTTCA-3’；

2MTBC-F 5’-ATGCCCAAGAGAAGCGAATACAGGCAA-3’；

MTBC-R 5’-CTATTGCTGCGGTGCGGGCTTCAA-3’；

3MT-F 5’-GCGCAGCTGCCGGATGTCAAC-3’；

MT-R 5’-CGCCGGCAGCCTCACGAAATG-3’；

4PncA-F 5’-CTCAGCTGGTCATGTTCCCGAT-3’；

PncA-R 5’-CGGTGTGCCGGAGAAGCCG-3’；

5RD1-F 5’-AAGCGGTTGCCGCCGACCGACC-3’；

RD1-R 5’-GAGGCGATCTGGCGGTTTGGGG-3’。

3. a PCR method of differentiating Mycobacterium bovis is characterized in that, is template with the sample total DNA, utilizes claim 1 or 2 described primers to carry out pcr amplification respectively, and reaction finishes the back according to the agarose gel electrophoresis result of determination.

4. method as claimed in claim 3 also comprises: when bacterium to be measured is unknown bacterium, be template with the sample total DNA, utilize described the 1st～5 pair of combination of primers of claim 2, carry out pcr amplification respectively; Or

When bacterium to be measured is mycobacterium species, be template with the sample total DNA, utilize described the 2nd～5 pair of combination of primers of claim 2, carry out pcr amplification respectively; Or

When bacterium to be measured is the mycobacterium tuberculosis complex bacterium, be template with the sample total DNA, utilize described the 3rd～5 pair of combination of primers of claim 2, carry out pcr amplification respectively; Or

When bacterium to be measured is mycobacterium tuberculosis, be template with the sample total DNA, utilize described the 4th～5 pair of combination of primers of claim 2, carry out pcr amplification respectively; Or

When bacterium to be measured is the Mycobacterium bovis bacterium, be template with the sample total DNA, utilize described the 5th pair of primer of claim 2, carry out pcr amplification; Reaction finishes the back according to the agarose gel electrophoresis result of determination.

5. like claim 3 or 4 described PCR methods, it is characterized in that,

The response procedures that contains the right PCR of 16SrRNA, MTBC, PncA primer is: 94 ℃ of sex change, 5min in advance, and again through 94 ℃ of sex change 40s, 60 ℃ of annealing 40s, 72 ℃ are extended 40s, 31 circulations, last 72 ℃ are extended 10min; Or

The response procedures that contains the right PCR of MT primer is: 94 ℃ of preparatory sex change, 5min, and again through 94 ℃ of sex change 40s, 61 ℃ of annealing 40s, 72 ℃ are extended 40s, 31 circulations, last 72 ℃ are extended 10min; Or

The response procedures that contains the right PCR of RD1 primer is: 94 ℃ of preparatory sex change, 5min, and again through 94 ℃ of sex change 40s, 62 ℃ of annealing 40s, 72 ℃ are extended 40s, 31 circulations, last 72 ℃ are extended 10min.

6. PCR method as claimed in claim 5 is characterized in that, said pcr amplification reaction system, and wherein the specific configuration of 20 μ l reaction solutions is:

7. like claim 3 or 4 described methods, it is characterized in that sample total DNA is from isolating microorganism resource or animal tissues.

8. test kit of differentiating Mycobacterium bovis, it comprises, claim 1 or 2 described combination of primers.

9. claim 1 or 2 described primers are to being used for detecting and differentiating the purposes of Mycobacterium bovis reagent in preparation.

10. described primer of claim 1～2 or the described method of claim 3～4 application in preparation prevention tuberculosis vaccine.

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