CN108165565A

CN108165565A - Mycobacterium tuberculosis H37Rv encoding gene and its application

Info

Publication number: CN108165565A
Application number: CN201711251282.2A
Authority: CN
Inventors: 徐平; 张瑶; 王富强; 孙金帅; 武舒佳; 常蕾
Original assignee: BEIJING PROTEOME RESEARCH CENTER
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2017-12-01
Filing date: 2017-12-01
Publication date: 2018-06-15
Anticipated expiration: 2037-12-01
Also published as: CN108165565B

Abstract

The present invention relates to a kind of Mycobacterium tuberculosis H37Rv encoding genes, can be used as the standard gene of mycobacterium tuberculosis complex Molecular Identification, for the Molecular Identification and clinical detection of mycobacterium tuberculosis complex.

Description

Mycobacterium tuberculosis H37Rv encoding gene and its application

Technical field

The present invention relates to genetic test fields, and in particular to pathogen species are identified.

Background technology

Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) is to cause mankind pathogen lungy. Each organ of whole body can be invaded, but using pulmonary tuberculosis to be most common.Tuberculosis is infectious disease particularly important so far, the serious threat mankind Life and health.According to WHO, every year there are about 8,000,000 new cases generation, at least 3,000,000 people die of the disease.The clinical bacterium of MTB Strain hardly possible cultivates, is slow-growing and other mycobacteria energy cross-infections, tuberculosis and other respiratory tract infection symptom difficulties are distinguished etc. Feature brings great difficulty to clinical quick diagnosis and treatment.Therefore establish quick, accurate, special, sensitive, cheap knot Core disease detection method is the prerequisite and clinical labororatory's mycobacteria detection faces of effective treatment, control tuberculosis sprawling The new challenge faced and new task.

Mycobacterium tuberculosis complex (Mycobacterium tuberculosis complex, MTBC), including M.tuberculosis、M.africanum、M.orygis、M.bovis、M.microti、M.canettii、M.caprae、 The mycobacterias monoid such as M.pinnipedii, M.suricattae, M.mungi, these species can cause people and other life Body tuberculosis.Following three classes are broadly divided into the identification method of MTBC both at home and abroad at present：Traditional isolated culture；Molecular level is examined It surveys (IS6110, restriction fragment length polymorphism analysis, multidigit point variable number repeated fragment polymorphism analysis etc.)；Thalline into Divide (aliphatic acid, mycolic acid) chromatogram analysis method.Though three classes method has the advantages of respective, also there is shortcoming, such as pass System is separately cultured period length, and with thalline can to cultivate rate low；Molecular level detection at present is in terms of specificity, sensitivity and simplicity It is still poorer；Drive member specificity analysis cost is higher, complicated for operation.

MTB H37Rv completed genome sequencing in 1998, were the earliest MTB bacterial strains for completing full figure sequencing.Since then, respectively State researchers are tactful based on algorithm optimization, annotating software update, transcription group and proteomics etc., always in perfect, benefit Fill H37Rv gene annotation databases.However, since MTB belongs to prokaryotes, due to prokaryotic gene group remarking technology in itself Deficiency, in genome annotation still there may be annotation mistake (excessively annotation, genetic borders mistake and ORF starting, stop bit Point mistake, alternative splicing, ribosomes displacement, leakage annotation), to deeply, accurately parse biological mechanism bring puzzlement.To solve This problem, protein gene group (Proteogenomics) though have been used for the correction that H37Rv has annotated gene, however, high Ratio false positive, routine techniques are difficult to annotation predictive genes, new gene verification, new gene functional analysis and its application etc. The problem that the field is faced.Generally speaking, traditional mycobacterium tuberculosis complex (MTBC) identification strategy has period length, step Suddenly the defects of cumbersome, specific and sensitivity is not high.H37Rv full-length genomes are annotated again further to improve, find H37Rv It is middle to omit annotation gene, it is ensured that H37Rv full-length genomes omit annotation gene and its application technology in MTBC Molecular Identifications obtains To effective protection, it is imperative to develop and use H37Rv new genes fast accurate mirror method for distinguishing in MTBC monoids.

Invention content

It is an object of the present invention to provide a kind of new encoding gene of Mycobacterium tuberculosis H37Rv, which is H37Rv Leakage annotation encoding gene Rv2003 (- | 2249260-2249457 |), it can be used as mycobacterium tuberculosis complex bar code molecule Label, for detecting mycobacterium tuberculosis complex, sequence is as shown in SEQ ID NO.1.

Other objects of the present invention include providing Specific PCR primers and the offer that can be used for expanding above-mentioned encoding gene It is a kind of to detect or identify the detection method that whether there is mycobacterium tuberculosis complex in sample；The present invention also provides with above-mentioned volume The application of the detection kit and said gene of code gene-correlation.

According to an aspect of the present invention, by comparing protein gene group investigative technique, it was found that one in H37Rv It is difficult to by the albumen coded sequence of predictive genes software discovery, which can be effectively by MTBC and the other species differentiations belonged to It comes.The gene is the omission annotation base of a mycobacterium tuberculosis (Mycobacterium tuberculosis H37Rv) Cause, i.e. Rv2003 (- | 2249260-2249457 |), it is similar to M.tuberculosis Bir 105 after NCBI-BLASTP Property be 100%, M.tuberculosis J09701920 similitudes be 98%, M.canettii RefSeq (WP_ 041179674.1) similitude is 97%, other species similarities are below 77%, and functional annotation albumen is Phosphoenolpyruvate synthase.It is studied through comparative genomics and finds that the gene order can be by mycobacterium tuberculosis Other kinds of compound group's (MTBC) bacterial strain and Mycobacterium, which distinguish, to be come.

Specifically, design can realize that specific amplification draws to the Rv2003 of MTBC (- | 2249260-2249457 |) gene Object, primer as proposed by the invention, primer sequence are：

F:5’-CCAACCATCACTGTCAGC-3’；

R:5’-AGCAACAATCGGCGTTC-3’。

The difference of the presence or absence of gene DNA sequence PCR product in sample to be tested or DNA sequence dna, can be quickly accurate True identification of M TBC.

According to another aspect of the present invention, based on the new standard code gene of above-mentioned Mycobacterium tuberculosis H37Rv, this hair The bright method for specifically establishing detection or identifying mycobacterium tuberculosis complex, step are as follows：

(1) the separation and Extraction genomic DNA from sample to be tested；

(2) DNA obtained using step (1) carries out PCR amplification as template using following primer：

F:5’-CCAACCATCACTGTCAGC-3’(SEQ ID NO.4)；

R:5’-AGCAACAATCGGCGTTC-3’(SEQ ID NO.5)。

(3) DNA product obtained to step (2) amplification carries out gel electrophoresis analysis or is sequenced；

(4) result of step (3) and bar code gene Rv2003 (- | 2249260-2249457 |) are compared, if Homology is more than 99%, and judgement sample to be tested contains mycobacterium tuberculosis complex.

Further, above-mentioned detection method according to DNA bar code principle, tentatively carries out electrophoretic analysis, such as to PCR product Fruit strain to be tested does not have target stripe, and it is not MTBC to illustrate the bacterial strain；If there is band, then can further sequence verification, will survey The standard sequence that sequence obtains series and the Rv2003 of H37Rv (- | 2249260-2249457 |) carries out Homology search and comparison, obtains The similitude between sequence is obtained, if sequence homology is more than 99%, you can judgement bacterial strain may be MTBC；According to identification bacterium to be identified The DNA bar code sequence of strain and standard sequence cluster situation are common to distinguish MTBC families and non-tuberculous mycobacteria, respiratory tract Pathogen and common respiratory tract virus.

The detection method can be used to the strain idenfication research to mycobacterium tuberculosis complex, it can also be used to clinical quick It examines.Sample to be tested can be from H37Rv bacterial strains, other MTBC, non-tuberculous mycobacteria, respiratory tract encountered pathogenic bacteria, breathing Road common virus bacterial strain；Either directly using tuberculosis and other respiratory tract patient sputums, saliva or blood.

Based on the above method, the present invention also provides detection kit, and kit containers are built-in to detect knot The reagent of standard code gene new core mycobacteria H37Rv, what is concurrently provided can be through governmental drug management organization Manufacture, use and sales informations audit, in relation to drug or biological products.For example, after using PCR amplification, sample is directly detected The reagent of Rv2003 in product (- | 2249260-2249457 |) gene, such as amplimer, dNTP can be contained, reacted for PCR Archaeal dna polymerase and its buffer solution, endonuclease reaction and/or reagent needed for sequencing reaction etc. it is one or more.Art technology Personnel are it is known that more than component is only illustrative, for example, above-mentioned Specific PCR primers, the use may be used in the primer The enzyme of PCR amplification is can be used in the archaeal dna polymerase of PCR reactions.The detection of the encoding gene of the present invention can also be with integrated The mode of such as genetic chip provide.

Advantageous effect：The present invention provides one kind to be used as mycobacterium tuberculosis complex (Mycobacterium Tuberculosis complex, MTBC) Molecular Identification standard gene and method for identifying molecules, the gene can effectively by MTBC comes with the other species differentiations belonged to, and existing mycobacterium tuberculosis complex is overcome using the identification method of the gene The shortcomings of design of primers multiplicity in qualification process, result poor repeatability, has the characteristics that general, easy amplification, easily compares, can Accurately to identify to come from the close other mycobacterias of affiliation or other respiratory tract infection germs by the monoid, it is Tuberculosis epidemiological survey and clinical tuberculosis patient quick diagnosis differentiate the strong technological means of offer and research tool.

Description of the drawings

Fig. 1：Support the peptide spectrum matching evidence of the newly encoded gene found；

Fig. 2：Section of synthesized peptide mass spectrogram is compared with former identification peptide fragment mass spectrogram；

Fig. 3：Peptide fragment is located the protein sequence corresponding diagram of region ORF codings；It is identified for proteomics underscore part And it is synthesized the peptide fragment of peptide fragment verification；

Fig. 4：Rv2003 (- | 2249260-2249457 |) standard gene sequence homology compares；

Fig. 5：Protein sequence BLASTP knots corresponding to H37Rv bacterial strains Rv2003 (- | 2249260-2249457 |) gene Fruit；

Fig. 6：Rv2003 (- | 2249260-2249457 |) specific primer PCR amplified production agarose gel electrophoresis result；

Wherein, each Lane Sample information is shown in Table 1.

Fig. 7：Rv2003 (- | 2249260-2249457 |) gene PCR expands sequencing result and standard sequence compares.

Specific embodiment

The present invention will be further described With reference to embodiment, but does not limit scope of the invention as claimed. Agents useful for same of the present invention is commercially available.

Embodiment 1：Find the leakage annotation encoding gene of H37Rv strain gene groups

The high covering protein group verification of 1.1 pairs of H37Rv strain gene groups

The depth that protein group has been carried out to H37Rv bacterial strains using high covering protein technique covers research.It is based on Tuberculosis (20160307) database has carried out its genome using 3 engines of pFind the verification of annotation encoding gene. In order to find new protein-coding region, we are based on protein gene omics technology, and H37Rv is sent out in NCBI with pAnno softwares Full-length genome (NC_000962.3) file of table carries out six reading frame data base interpretations, and using this database to spectra count According to the identification for having carried out new peptide fragment and novel protein.In order to reduce false positive rate, we have used 3 during data filtering Kind separately estimates the filter method of classification FDR to having annotated peptide fragment and new peptide fragment, is S-FDR, T-FDR I and T-FDR respectively II。

Through data analysis, we identify 3238 H37Rv and have annotated gene altogether, coverage be up to the 80% of the bacterial strain with On, this is to report maximum H37Rv protein spectrum data so far.In addition, after being filtered through 3 kinds of FDR≤1, we obtain new peptide fragment. In order to further ensure that new peptide fragment quality, the spectrogram corresponding to our new peptide fragments remaining to above-mentioned filtering has carried out spectrogram quality Screening, finally remains the high-quality peptide fragment of some spectrograms.For further investigate the higher peptide fragment of these spectrogram quality not by Caused by having annotated peptide fragment and single amino acids mutation occurs, We conducted amino acid mutation verifications, it is ensured that these new peptide fragments are H37Rv newly identifies peptide fragment.

The coding albumen and database authentication of 1.2 couples of Rv2003 (- | 2249260-2249457 |) gene

After excessively high covering protein group verification, it has been found that some doubtful new leakage annotation peptide fragments, it can to above-mentioned height Doubtful new peptide fragment progress peptide fragment synthesis verification is believed to obtain, according to new peptide fragment original spectrum and peptide fragment synthesis spectrum similarity >=0.8 conduct of marking Similarity threshold after marking is screened, has several peptide fragments by verification, corresponding to new open reading frame (Open Reading Frame, ORF), i.e., the potential leakage annotation gene of current H37Rv bacterial strains.

Wherein, it has been found that new leakage annotation gene Rv2003 (- | 2249260-2249457 |), compare through BLASTP, with 105 similitudes of M.tuberculosis Bir are that 100%, M.tuberculosis J09701920 similitudes are 98%, The similitude of M.canettii RefSeq (WP_041179674.1) is 97%, and other species similarities are below 77%, work( It is Phosphoenolpyruvate synthase that albumen, which can be annotated,.We detect peptide fragment MGITSVSVHSGAIAATPGSVAAAER (SEQ ID NO.6), and corresponding to new gene Rv2003 (- | 2249260-2249457 |), as shown in Figure 1, spectrogram quality is fine, there are continuous 5 b/y ion continuous couplings, miscellaneous peak signal is relatively low, as a result very credible.

Further to confirm this qualification result, we newly identify that the amino acid sequence chemistry of peptide fragment is synthesized according to us The peptide fragment, and produce using above-mentioned mass spectral analysis condition the two level spectrogram of the section of synthesized peptide.

The high energy collision MS that we generate section of synthesized peptide₂It is verified, level-one parent ion and two level daughter ion are equal Meet theoretical value, show that the peptide section sequence that we synthesize is correct；On this basis, we are had checked according to large-scale protein by hand The MS of the section of synthesized peptide of new peptide section sequence that matter group data authentication arrives₂With the new peptide fragment spectrogram of Large scale identification, the two is almost The one cosin values for showing the acquisition of daughter ion similitude are 0.87, it was demonstrated that the correct nothing of new peptide fragment that we identify from H37Rv Accidentally.(Fig. 2).

After the sequence for confirming above-mentioned leakage annotation peptide fragment, according to the gene location where above-mentioned peptide fragment, with previous termination The region that codon and the latter terminator codon include is boundary, obtains including the open reading frame of above-mentioned new leakage annotation peptide fragment (ORF) DNA sequence dna, as shown in SEQ ID NO.2.

TGAGCGCTCCGTGGGACAGGCCTACCACATCTGGCGCAGCGCGATCTGAGAACGCCGAAAGGAAAACCGATGCCAAC CATCACTGTCAGCAGCACATCGTCGCTGTGTGGTCAAGCGCTCTCGGGCAACCCGACTTTCGCCGAGCATCTGGTCC GGATGGGAATCACCTCAGTGTCGGTCCATTCGGGCGCGATTGCTGCTACCCCGGGGTCGGTCGCGGCCGCCGAACGC CGATTGTTGCTGGAATCAGCTCGCGGTGACGCCTGA(SEQ ID NO.2)

Open reading frame coding is as shown in Figure 3 with the correspondence of amino acid sequence.

Further translation verification, finds true gene order (SEQ ID NO.1) from above-mentioned open reading frame DNA (SEQ ID NO.2) inATGStart, common 198bp, 65 amino acid of coding, theoretical molecular weight 6.51kDa, as Rv2003 (- | 2249260-2249457 |) gene.

ATGCCAACCATCACTGTCAGCAGCACATCGTCGCTGTGTGGTCAAGCGCTCTCGGGCAACCCGACTTTCGCCGAGCA TCTGGTCCGGATGGGAATCACCTCAGTGTCGGTCCATTCGGGCGCGATTGCTGCTACCCCGGGGTCGGTCGCGGCCG CCGAACGCCGATTGTTGCTGGAATCAGCTCGCGGTGACGCCTGA(SEQID NO.1)

The gene theory coded product amino acid sequence is as shown in SEQ ID NO.3：

MPTITVSSTSSLCGQALSGNPTFAEHLVRMGITSVSVHSGAIAATPGSVAAAERRLLLESARGDA(SEQ ID NO.3)

NCBI-BLASTP analyses are carried out to the amino acid sequence of gene encoding production theoretical shown in the SEQ ID NO.3, with 105 similitudes of M.tuberculosis Bir are that 100%, M.tuberculosis J09701920 similitudes are 98%, The similitude of M.canettii RefSeq (WP_041179674.1) is 97%, and other species similarities are below 77%, work( It is Phosphoenolpyruvate synthase that albumen, which can be annotated, (see Fig. 4).Show Rv2003 that we detect (- | 2249260-2249457 |) gene outcome is missed annotation in H37Rv bacterial strain databases.

The DNA sequence dna of the Rv2003 (- | 2249260-2249457 |) gene is compared genome local by us BLAST is analyzed, as shown in Figure 5, the results showed that Rv2003 (- | 2249260-2249457 |) gene order belongs to MTBC families spy Specific gene, the sequence for not having homology higher in other species, this shows the Rv2003 that we have found in H37Rv bacterial strains (- | 2249260-2249457 |) gene order has preferable sequence-specific, MTBC and it can will belong to interior other mycobacterias And other Bacteria infecting respiratories distinguish.

Embodiment 2：The method for establishing the compound groups of identification of M TBC

(1) primer is designed：

Based on the CDS sequences of Rv2003 (- | 2249260-2249457 |) gene as shown in SEQ ID NO.1, use Oligo7.0 devises PCR primer, and primer sequence is as follows：

F:5’-CCAACCATCACTGTCAGC-3’(SEQ ID NO.4)；

R:5’-AGCAACAATCGGCGTTC-3’(SEQ ID NO.5)

Above-mentioned primer is as follows with the position relationship of Rv2003 (- | 2249260-2249457 |) gene, wherein primer Corresponding position subscript list is crossed.

ATGCCAACCATCACTGTCAGCAGCACATCGTCGCTGTGTGGTCAAGCGCTCTCGGGCAACCCGACTTTC GCCGAGCATCTGGTCCGGATGGGAATCACCTCAGTGTCGGTCCATTCGGGCGCGATTGCTGCTACCCCGGGGTCGGT CGCGGCCGCCGAACGCCGATTGTTGCTGGAATCAGCTCGCGGTGACGCCTGA

(SEQ ID NO.1)

(2) total DNA of strain to be tested of the extraction including M.tuberculosis H37Rv, 40 plants of Mycobacterium marks For quasi- bacterial strain by Chinese medicine bacteria culture preservation administrative center (CMCC) preservation, remaining 16 plants of non-tuberculous mycobacteria is Chinese 309 hospital clinical separation strains of people's liberation army have completed strain 16SRNA gene sequencing, comparison and NCBI sequences and have submitted work, Strain to be tested is as shown in table 1：

The related strain that table 1. is selected

(3) amplification of DNA fragments carries out polymerase chain (PCR) and reacts, and above-mentioned F/R primers used are expanded.

PCR system (25 μ L) is ddH₂O (9.5 μ L), 2XTaq PCR MasterMix (TIANGEN, 12.5 μ L) primers F (10 μM, 1 μ L), primer R (10 μM, 1 μ L), DNA profiling (1 μ L)；

Amplification program：94 DEG C of pre-degeneration 3min, 94 DEG C denaturation 30s, 58 DEG C annealing 30s, 72 DEG C extension 1min, 35 follow Ring, 72 DEG C of extension 5min.

(4) amplified production electrophoresis detection, the electrophoresis detection in Ago-Gel, 1 × TBE electrophoresis liquids.As a result such as Fig. 6 institutes Show, MTBC and control group amplified band occur, and practical amplification is consistent with expection at 170bp, and specificity is 98.3%.

(5) in order to further verify the sequence of the DNA of amplification, we have carried out extension increasing sequence sequencing and and former sequence ratio Compared with as shown in fig. 7, result is consistent completely with expection, sequence is correct, this further demonstrates depositing for new leakage annotation gene .

This shows that the method that the compound group's identifications of MTBC are carried out based on Rv2003 (- | 2249260-2249457 |) gene is true Reliably.

SEQUENCE LISTING

<110>Beijing Proteome Research Center

<120>Mycobacterium tuberculosis H37Rv encoding gene and its application

<130> BJ1936-17P121796

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 198

<212> DNA

<213> Artificial

<220>

<223>Mycobacterium tuberculosis H37Rv encoding gene Rv2003 (- | 2249260-2249457 |)

<400> 1

atgccaacca tcactgtcag cagcacatcg tcgctgtgtg gtcaagcgct ctcgggcaac 60

ccgactttcg ccgagcatct ggtccggatg ggaatcacct cagtgtcggt ccattcgggc 120

gcgattgctg ctaccccggg gtcggtcgcg gccgccgaac gccgattgtt gctggaatca 180

gctcgcggtg acgcctga 198

<210> 2

<211> 267

<212> DNA

<213> Artificial

<220>

<223>Include the open reading frame DNA sequence dna of leakage annotation peptide fragment

<400> 2

tgagcgctcc gtgggacagg cctaccacat ctggcgcagc gcgatctgag aacgccgaaa 60

ggaaaaccga tgccaaccat cactgtcagc agcacatcgt cgctgtgtgg tcaagcgctc 120

tcgggcaacc cgactttcgc cgagcatctg gtccggatgg gaatcacctc agtgtcggtc 180

cattcgggcg cgattgctgc taccccgggg tcggtcgcgg ccgccgaacg ccgattgttg 240

ctggaatcag ctcgcggtga cgcctga 267

<210> 3

<211> 65

<212> PRT

<213> Artificial

<220>

<223>Rv2003 (- | 2249260-2249457 |) gene theory coded product amino acid sequence

<400> 3

Met Pro Thr Ile Thr Val Ser Ser Thr Ser Ser Leu Cys Gly Gln Ala

1 5 10 15

Leu Ser Gly Asn Pro Thr Phe Ala Glu His Leu Val Arg Met Gly Ile

20 25 30

Thr Ser Val Ser Val His Ser Gly Ala Ile Ala Ala Thr Pro Gly Ser

35 40 45

Val Ala Ala Ala Glu Arg Arg Leu Leu Leu Glu Ser Ala Arg Gly Asp

50 55 60

Ala

65

<210> 4

<211> 18

<212> DNA

<213> Artificial

<220>

<223>F primer sequences

<400> 4

ccaaccatca ctgtcagc 18

<210> 5

<211> 17

<212> DNA

<213> Artificial

<220>

<223>R primer sequences

<400> 5

agcaacaatc ggcgttc 17

<210> 6

<211> 25

<212> PRT

<213> Artificial

<220>

<223>Leakage annotation peptide fragment

<400> 6

Met Gly Ile Thr Ser Val Ser Val His Ser Gly Ala Ile Ala Ala Thr

1 5 10 15

Pro Gly Ser Val Ala Ala Ala Glu Arg

20 25

Claims

1. a kind of Mycobacterium tuberculosis H37Rv encoding gene, the nucleotide sequence such as SEQ ID NO.1 institutes of the encoding gene Show.

2. Mycobacterium tuberculosis H37Rv encoding gene described in claim 1, it is characterised in that the gene code such as SEQ ID Amino acid shown in NO.3 sequences.

3. a kind of bar code molecular labeling, as detecting and/or identifying mycobacterium tuberculosis complex, it includes examined as standard The Mycobacterium tuberculosis H37Rv encoding gene described in claim 1 of cls gene.

A 4. species-specific PCR primers, for expanding Mycobacterium tuberculosis H37Rv encoding gene described in claim 1.

5. the PCT primers described in claim 4, which is characterized in that the sequence of the primer is：

F:5’-CCAACCATCACTGTCAGC-3’；

R:5’-AGCAACAATCGGCGTTC-3’。

6. a kind of detection method of mycobacterium tuberculosis complex identification, includes the following steps：

(1) the separation and Extraction genomic DNA from sample to be tested；

(2) DNA obtained using step (1) adds in amplimer as template, carries out PCR；

(4) result of step (3) is compared with encoding gene described in claim 1, it is to be measured according to its homologous sex determination It whether there is mycobacterium tuberculosis complex in sample.

7. the amplimer sequence described in the detection method described in claim 6, wherein step (2) is：

F:5’-CCAACCATCACTGTCAGC-3’；

R:5’-AGCAACAATCGGCGTTC-3’。

8. the detection method described in claim 6, wherein in step (4), if homology is more than 99%, test sample is treated in judgement Contain mycobacterium tuberculosis complex in product.

9. a kind of detection kit, will comprising Mycobacterium tuberculosis H37Rv encoding gene described in claim 1 and/or right Seek the Specific PCR primers described in 4.

10. Mycobacterium tuberculosis H37Rv encoding gene described in claim 1 is in tuberculosis epidemiological survey and/or clinical knot Application in core patient quick diagnosis and discriminating.