CN113481311B

CN113481311B - SNP molecular marker for identifying Brucella vaccine strain M5 and application thereof

Info

Publication number: CN113481311B
Application number: CN202111041179.1A
Authority: CN
Inventors: 姜海; 张雯; 朴东日; 杨晓雯; 田国忠; 赵鸿雁; 范玉
Original assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Current assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Priority date: 2021-09-07
Filing date: 2021-09-07
Publication date: 2021-12-31
Anticipated expiration: 2041-09-07
Also published as: CN113481311A

Abstract

The invention relates to the technical field of molecular markers, in particular to an SNP molecular marker for identifying a Brucella vaccine strain M5 and application thereof. The SNP molecular marker provided by the invention comprises a nucleotide sequence with the 37 th polymorphism of A/C, which is positioned in the sequence shown as SEQ ID NO.1, wherein the polymorphic site of the SNP molecular marker is A and corresponds to Brucella vaccine strain M5, and the polymorphic site is C and corresponds to wild strain or other Brucella vaccine strains. The SNP molecular marker, the detection primer and the probe thereof can realize simple and efficient identification of the Brucella vaccine strain M5, the wild strain and other Brucella vaccine strains, have high accuracy, can be applied to pathogen monitoring and epidemiological analysis of Brucella diseases, and provide accurate technical support for prevention and control of Brucella diseases.

Description

SNP molecular marker for identifying Brucella vaccine strain M5 and application thereof

Technical Field

The invention relates to the technical field of molecular markers, in particular to an SNP molecular marker for identifying a Brucella vaccine strain M5 and application thereof.

Background

Brucella is a parasitic gram-negative coccobacillus in cells, causing brucellosis (shortly called brucellosis). Brucella is divided into 6 species for a total of 19 biotypes by 2006, including 8 biotypes of a bovine species (Brucella abortus), 3 biotypes of a ovine species (Brucella melitensis), 5 biotypes of a porcine species (Brucella suis), 1 each of a canine species (Brucella canis), an epididymis ovis species (Brucella ovis), and a desert forest rat species (Brucella neotomae). Currently, the number of brucella is increased by 5 new species: brucella ceti (isolated from whales and dolphins), Brucella pinipendialis (isolated from finpods), Brucella microti (isolated from mice), Brucella inopinata (isolated from blood and wound exudate from breast transplant patients) and Brucella papionis (isolated from baboons). The G + C content of each biological type of the Brucella is 55-59%, the DNA is highly homologous, the homology is over 90%, and the genome size and the composition are similar.

The immune livestock can not only protect the livestock from being infected by the Brucella, but also indirectly protect the crowd. Brucella melitensis strain No.5 (M5 vaccine) is a vaccine for livestock commonly used for immunizing livestock. The vaccine is used for immunizing sheep flocks (sheep and goats), the immunization method is subcutaneous injection or aerosol immunization, the use is convenient, the protection period is 1 year, and the effect is good. The immunity of the cattle flora is usually good by using live vaccine of the Brucella 19 strain (subcutaneous injection) and live vaccine of the Brucella 2 strain (S2 vaccine).

The differential diagnosis of brucella wild strains and vaccine strains is always a hotspot of brucella research. In animal epidemiological investigations and epidemic situations, the identification of natural infection and vaccine infection by serological detection is often not specific, and therefore the search for specific identification sites from the viewpoint of molecular biology is imminent. The comparative genomics can not only perform the comparison of the whole genome and the phylogenetic evolutionary relationship analysis, but also perform the research of bacterial genome polymorphism, thereby revealing the potential functions of genes, and clarifying the species evolutionary relationship and the internal structure of the genome. The comparative genomics research of the Brucella not only accelerates the discovery of new functional genes and main pathogenic difference genes, but also provides an important technical support for the differential diagnosis of the infection of vaccine strains and wild strains in clinical practice. With the development of the second-generation sequencing technology, the whole genome sequencing of common vaccine strains and parent strains is completed, and the difference of the vaccine strains and wild strains at the genome level is analyzed by utilizing comparative genomics, so that a novel method for differential diagnosis of the vaccine strains and the wild strains is developed. Compared genomics research on the sheep virulent strain M28-12 and sheep vaccine strains M5 and M111 shows that total 1370 single nucleotide polymorphisms of M5, M28-12 and M111, wherein 89 single nucleotide polymorphisms are from M5, Mlll and M28-12, and the polynucleotide polymorphic sites are probably from mutation of the vaccine strains. The discovery of these mutation sites has important implications for designing new, safer vaccines.

With the development of sequencing technology, bacterial genome sequencing and whole genome comparison provide a brand-new method for grouping and clustering population structures based on analysis of bacterial whole genomes. However, this method has the disadvantages of being costly and highly dependent on the sequencer and cannot be applied to infrastructure lacking a sequencer. SNPs obtained based on pairwise comparison of bacterial genomes can only represent differences among strains, and whether the SNPs can be used for identification or typing needs to be evaluated experimentally. However, the number of SNPs obtained by comparison among bacterial genomes is hundreds, and the number of SNPs obtained by comparison among bacterial genomes is tens of thousands, so that the workload is very large if the SNPs are sequentially evaluated, and a new technology of multiple bacterial genome comparison is adopted to screen the SNP sites.

At present, a real-time fluorescent quantitative PCR (RT-PCR) method is widely applied to the aspects of brucella detection, brucella strain identification, vaccine strain and wild strain distinguishing and the like, and meets the requirements of various aspects such as inspection and quarantine, epidemiological investigation, human and animal epidemic diagnosis and the like.

The brucella vaccine strain M5 can cause human infection, and the antibody generated after the animal is inoculated with the M5 vaccine is difficult to be distinguished and diagnosed with the antibody generated by the naturally infected wild strain, thereby influencing the animal epidemic monitoring and the epidemic situation treatment. Therefore, the development of molecular markers for distinguishing and identifying the Brucella vaccine strain M5 and a simple, rapid, specific and low-cost RT-PCR identification method are of great significance.

Disclosure of Invention

The invention aims to provide an SNP molecular marker for identifying a Brucella vaccine strain M5 and a primer probe combination thereof. The invention also aims to provide the application of the SNP molecular marker and the primer probe combination thereof.

In order to achieve the aim, the invention carries out systematic analysis on the brucella whole genome information based on the minimum core group type technology for the first time. Specifically, Complete and Scaffold genomes disclosed by Brucella melitensis (136 strains) are downloaded from an NCBI public database, based on a Blast method, genome GCA _000348645.1 sequenced by an M5 vaccine strain stored in a Brucella disease control room of infectious diseases of the Chinese disease control center is used as a reference genome, and is sequentially compared with 135 genome of Brucella melitensis, and if the similarity is more than 60% and the compared sequence exceeds 70% of the original sequence, the gene is considered to be present in the genome. According to this analysis strategy, Core genes present in 135 strains of Brucella ovis were searched. And sequentially comparing the Core gene sequence with 135 genomes by using a Mummer program, searching SNP sites and constructing an SNP matrix.

Defining 5M 5 vaccine strains (two strains are from the sequence sequenced by brucellosis control room of infectious disease of Chinese disease control center, and three strains are from NCBI database) as Group 1 (vaccine Group); 131 Brucella wild strains (of 136, 131 are wild strains) and two non-M5 Brucella vaccine strains S2 (porcine species) and A19 (bovine species) were defined as Group 2. And (3) screening Single Nucleotide Polymorphism (SNP) sites which can distinguish two groups of strains based on the SNP matrix obtained in the last step and 119756 SNPs in total. SNP sites which can distinguish two groups of strains have the characteristics of no difference among the strains in the site group and difference among the groups. According to the above strategy, 4 SNP sites are screened out altogether. And further screening the 4 sites (because 1-3 SNPs exist in the sequence of the other three sites and the identification of the vaccine strain is interfered, only one site which has only one SNP except the target gene but no other SNPs is selected), finally obtaining the SNP molecular marker capable of accurately identifying the Brucella vaccine strain M5, and designing and screening primers and probes for detecting the SNP molecular marker.

Specifically, the invention provides the following technical scheme:

the invention provides a SNP molecular marker for identifying a Brucella vaccine strain M5, which comprises a nucleotide sequence with polymorphism A/C at the 37 th site of the sequence shown as SEQ ID NO. 1.

The polymorphic site of the SNP molecular marker is positioned at the 37 th site of the brucella MKPBFDOI-01277 hypothetic protein, and the polymorphism is A/C.

MKPBFDOI _01277 gene sequences of different strains of Brucella can be obtained through a database, wherein the MKPBFDOI _01277 gene sequence of Brucella vaccine strain M5 is shown in SEQ ID NO. 1.

The SNP molecular marker can be a DNA fragment with a sequence shown as SEQ ID NO. 1.

The polymorphic site of the SNP molecular marker is A and corresponds to Brucella vaccine strain M5, and the polymorphic site is C and corresponds to a wild strain or other vaccine strains.

Further, the invention provides a primer group for identifying the Brucella vaccine strain M5, and the nucleotide sequence of the primer group is shown as SEQ ID NO. 2-3.

SEQ ID NO.2：MKPBFDOI_01277-F:

5’-AGGCGTCGCAAATCTGGTAA-3’；

SEQ ID NO.3：MKPBFDOI_01277-R:

5’-AACTGGTAGAAGCGCGCCC-3’。

The invention also provides a probe set used by being matched with the primer set, and the nucleotide sequence of the probe set is shown in SEQ ID NO. 4-5.

SEQ ID NO.4：MKPBFDOI_01277-A:

5’- CTGAAAAATATAGCGAAGAC-3’；

SEQ ID NO.5：MKPBFDOI_01277-C:

5’-CTGAAAAATCTAGCGAAGA-3’。

The probes shown in SEQ ID NO.4-5 are matched with the A/C polymorphism sequence at the 37 th site of the MKPBFDOI _01277 gene (shown in SEQ ID NO. 1) respectively.

The probe described above is an MGB probe.

Preferably, the probe shown in SEQ ID NO.4 and the probe shown in SEQ ID NO.5 are labeled with different fluorophores. The fluorescent group can be selected from any one of FAM, VIC, HEX, CY5, TET, JOE, CY3, TAMRA and ROX.

As an embodiment of the present invention, the 5 'end of the probe shown in SEQ ID NO.4 is labeled with FAM and the 3' end is labeled with BHQ 1. The 5 'end of the probe shown in SEQ ID NO.5 is labeled with VIC, and the 3' end is labeled with BHQ 1.

The invention provides application of the SNP molecular marker or the detection reagent thereof, or the primer group or the probe group in identifying the Brucella vaccine strain M5.

The invention also provides application of the SNP molecular marker or the detection reagent thereof, or the primer group or the probe group in preparing a kit for identifying the Brucella vaccine strain M5.

The invention provides a kit for identifying a Brucella vaccine strain M5, which comprises a primer group shown in SEQ ID NO.2-3, or comprises a primer group shown in SEQ ID NO.2-3 and a probe group shown in SEQ ID NO. 4-5.

The kit described above may also include other reagents for RT-PCR detection, including but not limited to reaction buffer, dNTPs, DNA polymerase, ddH₂O, standard positive plasmid template, negative control and the like.

Specifically, the working procedure of the kit comprises the following steps:

(1) extracting the genome DNA of a sample to be detected;

(2) performing RT-PCR by using the genomic DNA as a template and using a primer group shown in SEQ ID NO.2-3 and a probe group shown in SEQ ID NO. 4-5;

(3) and judging whether the sample to be detected is the brucella vaccine strain M5 or not according to the amplification curve.

In the step (2), the reaction procedure of RT-PCR comprises: 5-10 minutes at 95 ℃; 95 ℃ for 10-30 seconds, 62 ℃ for 20-30 seconds, and 35-45 cycles.

Preferably, the reaction procedure of RT-PCR is: 10 minutes at 95 ℃; 95 ℃, 15 seconds, 62 ℃, 30 seconds, 40 cycles.

In the step (2), the concentration of the primer set represented by SEQ ID NO.2-3 in the RT-PCR reaction system is 300-350nM, and the concentration of the probe set represented by SEQ ID NO.4-5 is 200-250 nM.

In the step (3), if the primer group shown in SEQ ID NO.2-3 and the probe shown in SEQ ID NO.4 can obtain an amplification curve, the sample to be detected is the Brucella vaccine strain M5; if the primer group shown in SEQ ID NO.2-3 and the probe shown in SEQ ID NO.5 can obtain an amplification curve, the sample to be detected is a wild strain or other vaccine strains of Brucella.

The invention provides a method for identifying a brucella vaccine strain M5, which comprises the following steps:

(1) extracting the genome DNA of a sample to be detected;

(2) taking the genome DNA as a template, and respectively carrying out RT-PCR by using a primer group shown by SEQ ID NO.2-3, a probe shown by SEQ ID NO.4 and a probe shown by SEQ ID NO. 5;

The invention has the beneficial effects that:

(1) the invention carries out whole genome comparison analysis of the Brucella vaccine strain M5 and other vaccine strains and wild strains based on the minimum core component type technology, covers all published Chinese epidemic strains, international reference strains and international epidemic strains in an NCBI database, and effectively ensures high specificity and accuracy of the screened SNP sites.

(2) Based on the SNP molecular markers obtained by screening, a pair of primer pairs for identifying the Brucella vaccine strain M5 and the wild strain and a pair of MGB probes are developed, the probes can be mutually verified, and the primer pairs and the probe pairs can be used for efficiently and accurately identifying the Brucella vaccine strain M5, the wild strain and other Brucella vaccine strains.

(3) The SNP molecular marker, the detection primer pair and the probe pair thereof can carry out rapid synchronous detection and analysis on the Brucella vaccine strain M5, the wild strain and other Brucella vaccine strains, effectively improve the detection efficiency, realize rapid detection and typing of Brucella, simply, conveniently and efficiently identify the separated strain, have important significance for rapid identification and identification of Brucella, pathogen monitoring of Brucella and epidemiological analysis, and provide efficient technical support for prevention and control of Brucella.

Drawings

FIG. 1 shows the results of the primer and probe specificity evaluation verified by using the positive plasmid containing MKPBFDOI _01277 gene of Brucella vaccine strain M5 in example 1 of the present invention.

FIG. 2 shows the results of the primer and probe specificity evaluation in example 1 of the present invention using positive plasmid containing MKPBFDOI _01277 gene of Brucella wild strain.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the methods used in the following examples are conventional methods, and consumables and reagents used in the examples are commercially available.

Example 1 acquisition of core SNP molecular marker for identifying Brucella vaccine strain M5

1. Systematic analysis of brucella whole genome information by using minimum core component type method

(1) The Complete and Scaffold genomes published by Brucella melitensis (co. 136 strain) were downloaded from the GenBank database (http:// www.ncbi.nlm.nih.gov/genome /) of the National Center for Biotechnology Information (NCBI).

(2) Based on the Blast method, the gene of M5GCA _000348645.1 and the genome of 135 Brucella strains are sequentially aligned, if the similarity is more than 60% and the aligned sequence exceeds 70% of the original sequence, the gene is considered to be present in the genome. According to this analysis strategy, Core genes present in all strains were searched for, totaling 1291.

(3) And sequentially comparing the Core gene sequence with 134 genomes by using a Mummer program, searching SNP sites and constructing an SNP matrix. Defining 5M 5 vaccine strains as Group 1 (vaccine Group); the 131 Brucella wild strains were defined as Group 2. And screening SNP sites which can distinguish two groups of strains based on the SNP matrix obtained in the last step. SNP sites which can distinguish two groups of strains have the characteristics of no difference among the strains in the site group and difference among the groups. According to the strategy, 4 candidate target gene SNP loci are screened out in total.

(4) Designing primers and probes at the upstream and downstream of each candidate SNP site, carrying out amplification verification on different Brucella melitensis wild strains and two non-M5 Brucella melitensis vaccine strains S2 (pig species), A19 (cattle species) and M5 vaccine strains, and finally obtaining SNP molecular markers capable of accurately distinguishing the Brucella melitensis vaccine strain M5 from the different Brucella melitensis wild strains and two non-M5 Brucella melitensis vaccine strains S2 (pig species) and A19 (cattle species), wherein the SNP molecular markers are positioned at the 37 th site of Brucella MKPDOI _01277 gene (the sequence of the MKPBFDOI _01277 gene of the Brucella melitensis vaccine strain M5 is shown as SEQ ID NO. 1), the polymorphism is A/C, the polymorphism site is A, the polymorphism site corresponds to the Brucella strain M5, and the polymorphism site is C and corresponds to the wild Brucella melitensis or other vaccine strains.

2. Detection primer, probe and detection method of SNP molecular marker

The nucleotide sequences of the detection primers and the probes of the SNP molecular markers finally obtained by screening in the step 1 are shown as SEQ ID NO. 2-5:

SEQ ID NO.2：MKPBFDOI_01277-F:

5’-AGGCGTCGCAAATCTGGTAA-3’；

SEQ ID NO.3：MKPBFDOI_01277-R:

5’-AACTGGTAGAAGCGCGCCC-3’。

SEQ ID NO.4：MKPBFDOI_01277-A:

5’- FAM-CTGAAAAATATAGCGAAGAC-BHQ1-3’；

SEQ ID NO.5：MKPBFDOI_01277-C:

5’-VIC-CTGAAAAATCTAGCGAAGA-BHQ1-3’。

respectively taking a positive plasmid containing the MKPBFDOI _01277 gene of a Brucella vaccine strain M5 and a positive plasmid containing the MKPBFDOI _01277 gene of a Brucella wild strain as templates, and performing RT-PCR amplification by using primers and probes shown in SEQ ID NO. 2-5.

The 20. mu.l reaction system for RT-PCR was: 2 x RT-PCR Mix10 u l, primer and probe mixture (SEQ ID NO.2-3 shown primer and SEQ ID NO.4-5 shown probe) 2 u l, deionized water 6 u l, positive plasmid template DNA 2 u l. The final concentration of the primer shown in SEQ ID NO.2-3 in the reaction system is 300nM, and the final concentration of the probe shown in SEQ ID NO.4-5 in the reaction system is 200 nM.

The reaction conditions of RT-PCR were: 10 minutes at 95 ℃; 95 ℃, 15 seconds, 62 ℃, 30 seconds, 40 cycles.

The amplification curves of the positive plasmid template are shown in figure 1 and figure 2, and the detection primer and the probe of the SNP molecular marker can be used for respectively amplifying the Brucella vaccine strain M5 and the wild strain to obtain specific amplification curves. If the primer group shown in SEQ ID NO.2-3 and the probe shown in SEQ ID NO.4 can obtain an amplification curve, the sample to be detected is Brucella vaccine strain M5; if the primer group shown in SEQ ID NO.2-3 and the probe shown in SEQ ID NO.5 can obtain an amplification curve, the sample to be detected is the wild strain of Brucella melitensis.

Example 2 evaluation of specificity of SNP molecular marker detection

The SNP molecular markers and primer pairs and probe pairs (shown in Table 1) obtained by screening in example 1 are subjected to detection specificity evaluation by taking a plurality of vaccine strains such as S2 (pig species), A19 (cattle species) and the like as samples to be detected, wherein the SNP molecular markers and primer pairs and probe pairs have serological cross reaction with Brucella or have a serological cross reaction with Brucella, and the Brucella choleraesuis, the Brucella tularensis Escherichia coli O:157, Escherichia coli O:16, Yersinia enterocolitica O:9, Staphylococcus aureus and other Brucella wild strains are used as samples to be detected, and the specific method is as follows:

1. extraction of genomic DNA

The extraction of the genome DNA of all bacteria samples to be detected including Bartonella henselae, Vibrio cholerae, Francisella tularensis Escherichia coli O:157, Escherichia coli O:16, Yersinia enterocolitica O:9 and Staphylococcus aureus is carried out by adopting a bacterial genome DNA extraction kit of Beijing Tiangen Biochemical reagent Limited and strictly according to the instruction in the kit.

2、RT-PCR

Respectively taking the genomic DNA of each bacterium extracted in the step (1) as a template, and carrying out RT-PCR by using primers shown in SEQ ID NO.2-3 and probes shown in SEQ ID NO. 4-5.

The 20. mu.l reaction system for RT-PCR was: 2 μ l of 2 XRT-PCR Mix10 μ l, 2 μ l of primer and probe mixture (primers shown in SEQ ID NO.2-3 and probes shown in SEQ ID NO. 4-5), 6 μ l of deionized water, and 2 μ l of genomic DNA. The final concentration of the primer shown in SEQ ID NO.2-3 in the reaction system is 300nM, and the final concentration of the probe shown in SEQ ID NO.4-5 in the reaction system is 200 nM.

3. And analyzing the result according to the RT-PCR amplification curve.

The results show that the fluorescent signal of the amplification curve of the Brucella vaccine strain M5 is FAM, the corresponding SNP locus is A, the fluorescent signal of the amplification curve of the Brucella vaccine strain S2 (pig species) and A19 (cattle species) is VIC, the corresponding SNP locus is C, the fluorescent signal of the amplification curve of each standard strain and wild strain is VIC, and the corresponding SNP locus is C. No amplification is carried out on other non-Brucella samples, which shows that the SNP molecular marker, the detection primer and the probe thereof have higher specificity on Brucella.

Example 3 application of SNP molecular marker in identifying Brucella vaccine strain M5 and local Brucella strain

The SNP molecular marker obtained by screening in the embodiment 1, a primer (SEQ ID NO. 2-3) and a probe (SEQ ID NO. 4-5) thereof are utilized to distinguish and identify the Brucella vaccine strain M5, the Brucella standard strain, 120 locally separated Brucella wild strain and Brucella vaccine strain S2 (pig species) and A19 (cattle species), and the extraction of genome DNA and the RT-PCR method are the same as the embodiment 2.

The identification results are shown in tables 2, 3 and 4, the fluorescent signal of the amplification curve of brucella vaccine strain M5 is the Fluorescence (FAM) of the probe marker shown in SEQ ID No.4, the corresponding SNP site is a, the fluorescent signal of the amplification curve of each standard strain (the name of the standard strain is shown in table 4) is the fluorescence (VIC) of the probe marker shown in SEQ ID No.5, the corresponding SNP site is C, the fluorescent signal of the amplification curve of 120 wild strains is VIC, the corresponding SNP site is C, the fluorescent signals of the amplification curves of brucella vaccine strains S2 (porcine species) and a19 (bovine species) are VIC, and the corresponding SNP site is C.

The results show that the SNP molecular marker can realize the specific distinguishing and identification of the Brucella vaccine strain M5 and the local Brucella strain, and the specificity is 100%.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims

1. An application of an SNP molecular marker in the preparation of a kit for identifying a Brucella vaccine strain M5 and a non-Brucella vaccine strain M5 is characterized in that the SNP molecular marker comprises a nucleotide sequence with polymorphism A/C at the 37 th site of a sequence shown as SEQ ID NO. 1;

the polymorphic site of the SNP molecular marker is A and corresponds to a Brucella vaccine strain M5, and the polymorphic site is C and corresponds to a non-Brucella vaccine strain M5;

the non-Brucella vaccine strain M5 comprises: the standard strain is characterized by comprising a sheep seed 1 type standard strain 16M, a sheep seed 2 type standard strain 63/9, a sheep seed 3 type standard strain Ether, a cattle seed 1 type standard strain 544A, a cattle seed 2 type standard strain 86/8159, a cattle seed 3 type standard strain Tulya, a cattle seed 4 type standard strain 292, a cattle seed 5 type standard strain B3196, a cattle seed 6 type standard strain 870, a cattle seed 7 type standard strain 63/75, a cattle seed 9 type standard strain L68, a pig seed 1 type standard strain S, a pig seed 2 type standard strain Tholnson, a pig seed 3 type standard strain 686, a pig seed 4 type standard strain 40, a canine type standard strain RM6/66, a sarin-forest rat type standard strain 5K33, a sheep epididymis type standard strain 63/290, a vaccine strain S2, a vaccine strain A19 and a wild strain.

2. The application of a primer and probe combination in preparing a kit for identifying a Brucella vaccine strain M5 and a non-Brucella vaccine strain M5 is characterized in that the nucleotide sequence of the primer is shown as SEQ ID NO. 2-3;

the nucleotide sequence of the probe is shown in SEQ ID NO. 4-5; wherein the probe shown in SEQ ID NO.4 is used for detecting Brucella vaccine strain M5, and the probe shown in SEQ ID NO.5 is used for detecting non-Brucella vaccine strain M5;

3. A method for identifying non-disease diagnostic purposes of brucella vaccine strain M5, said method comprising the steps of:

(1) extracting the genome DNA of a sample to be detected;

(2) taking the genome DNA as a template, and carrying out RT-PCR by using primers shown in SEQ ID NO.2-3 and probes shown in SEQ ID NO. 4-5;

(3) judging whether the sample to be detected is a brucella vaccine strain M5 or not according to the amplification curve; if the primer group shown in SEQ ID NO.2-3 and the probe shown in SEQ ID NO.4 can obtain an amplification curve, the sample to be detected is Brucella vaccine strain M5; if the primer group shown in SEQ ID NO.2-3 and the probe shown in SEQ ID NO.5 can obtain an amplification curve, the sample to be detected is a non-Brucella vaccine strain M5;

4. The method of claim 3, wherein the reaction sequence of RT-PCR comprises: 5-10 minutes at 95 ℃; 95 ℃ for 10-30 seconds, 62 ℃ for 20-30 seconds, and 35-45 cycles.

5. The method for non-disease diagnosis in accordance with claim 3 or 4, wherein the concentration of the primer set represented by SEQ ID NO.2-3 is 300-350nM and the concentration of the probe set represented by SEQ ID NO.4-5 is 200-250nM in the reaction system of RT-PCR.