CN110343706A - Mycobacterium tuberculosis H37Rv encoding gene and its application - Google Patents

Abstract

The present invention relates to a kind of Mycobacterium tuberculosis H37Rv and its applications, more particularly to new leakage annotation encoding gene Rv0007A (+| 10966-11121 |), its standard gene that can be used as mycobacterium tuberculosis complex Molecular Identification, Molecular Identification and clinical detection for 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 identifies pathogen species.

Background technique

Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) is to cause mankind pathogen lungy. Each organ of whole body can be invaded, but is most common with pulmonary tuberculosis.Tuberculosis is infectious disease particularly important so far, seriously threatens the 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 The difficult culture of strain, slow growth, with other mycobacteria energy cross-infections, tuberculosis and the differentiation of other symptoms of respiratory tract infection difficulties 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 and new task faced.

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 inspection It surveys (IS6110, restriction fragment length polymorphism analysis, multidigit point variable number repeated fragment polymorphism analysis etc.)；Thallus at Divide (fatty acid, mycolic acid) chromatogram analysis method.Though three classes method has the advantages that respective, also there is shortcoming, such as passes System is separately cultured the period, and long with thallus can to cultivate rate low；Molecular level detection at present is in terms of specificity, sensitivity and simplicity It is still poorer；It is drive member specificity analysis higher cost, complicated for operation.

MTB H37Rv is the earliest MTB bacterial strain for completing full figure sequencing in completion genome sequencing in 1998.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 database.However, since MTB belongs to prokaryotes, due to prokaryotic gene group remarking technology 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 carry out annotation predictive genes, new gene verifying, 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, complex steps, specifically The defects of property and sensitivity is not high.H37Rv full-length genome is annotated again further to improve, finds to omit annotation in H37Rv Gene, it is ensured that H37Rv full-length genome omits annotation gene and its application technology in MTBC Molecular Identification is effectively protected, It is imperative to develop and use H37Rv new gene fast accurate mirror method for distinguishing in MTBC monoid.

Summary of the invention

It is an object of the present invention to provide a kind of encoding gene that Mycobacterium tuberculosis H37Rv is new, which is H37Rv Leakage annotation encoding gene Rv0007A (+| 10966-11121 |), it can be used as mycobacterium tuberculosis complex bar code molecule mark Note, for detecting mycobacterium tuberculosis complex, sequence is as shown in SEQ ID NO.1.

Other objects of the present invention include providing the 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 mycobacterium tuberculosis (Mycobacterium tuberculosis H37Rv) Cause, i.e. Rv0007A (+| 10966-11121 |), after NCBI-BLASTP, any sequence is arrived without comparing in database, is belonged to Unknown Function albumen.It is studied through comparative genomics and finds that the gene order can be by mycobacterium tuberculosis complex (MTBC) bacterial strain It distinguishes to come with other kinds of Mycobacterium.

Specifically, design can Rv0007A (+| 10966-11121 |) gene to MTBC realize specificity amplification primer, i.e., For primer proposed by the invention, primer sequence are as follows:

F:5'-TGTGCCCGTCACGACGTTCGGTGAG-3'；

R:5’-CTAAGGCCCCTCTTCGTCACCGGTT-3’。

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

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

F:5'-TGTGCCCGTCACGACGTTCGGTGAG-3'(SEQ ID NO.4)；

R:5’-CTAAGGCCCCTCTTCGTCACCGGTT-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 Rv0007A (+| 10966-11121 |) are compared, if together Source property is greater than 99%, determines that sample to be tested contains mycobacterium tuberculosis complex.

Further, above-mentioned detection method tentatively carries out electrophoretic analysis to PCR product, such as according to DNA bar code principle Fruit strain to be tested does not have target stripe, illustrates that the bacterial strain is not MTBC；If there is band, then can further sequence verification, will survey The standard sequence that sequence obtains series and the Rv0007A of H37Rv (+| 10966-11121 |) carries out Homology search and comparison, obtains sequence Similitude between column can determine that bacterial strain may be MTBC if sequence homology is greater than 99%；According to identification bacterial strain to be identified DNA bar code sequence and standard sequence cluster situation to distinguish MTBC family and non-tuberculous mycobacteria, respiratory tract encountered pathogenic Bacterium 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 strain, other MTBC, non-tuberculous mycobacteria, respiratory tract encountered pathogenic bacteria, breathing Road common virus bacterial strain；Either directly use 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 provided with to detect knot The reagent of core mycobacteria H37Rv new standard code gene, 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, directly detecting sample after using PCR amplification The reagent of Rv0007A in product (+| 10966-11121 |) gene, for example, containing amplimer, dNTP, for PCR reaction Reagent needed for archaeal dna polymerase and its buffer, endonuclease reaction and/or sequencing reaction etc. it is one or more.Those skilled in the art Member is it is known that the above component is only illustrative, for example, the primer can use above-mentioned Specific PCR primers, described is used for The archaeal dna polymerase of PCR reaction can be used for the enzyme of PCR amplification.The detection of encoding gene of the invention can also be with integrated Such as the mode of genetic chip provides.

The utility model has the advantages that 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, overcomes existing mycobacterium tuberculosis complex using the identification method of the gene The disadvantages of design of primers multiplicity in qualification process, result poor repeatability, has the characteristics that general, easy amplification, easily compares, can Come with accurately identifying the monoid from the close other mycobacterias of affiliation or other respiratory tract infection germs, is Tuberculosis epidemiological survey and clinical tuberculosis patient quick diagnosis identify offer strong technological means and research tool.

Detailed description of the invention

Fig. 1: the peptide spectrum matching evidence of the newly encoded gene of discovery is supported；

Fig. 2: section of synthesized peptide mass spectrogram and former identification peptide fragment mass spectrogram comparison；

Fig. 3: peptide fragment is located the protein sequence corresponding diagram of region ORF coding；Underscore part is proteomics identification And it is synthesized the peptide fragment of peptide fragment verifying；

Fig. 4: Rv0007A (+| 10966-11121 |) standard gene sequence homology compares；

Protein sequence BLASTP result corresponding to Fig. 5: H37Rv bacterial strain Rv0007A (+| 10966-11121 |) gene；

Fig. 6: Rv0007A (+| 10966-11121 |) specific primer PCR amplified production agarose gel electrophoresis results；

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

Fig. 7: Rv0007A (+| 10966-11121 |) 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: the leakage for finding H37Rv strain gene group annotates encoding gene

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

The depth covering research of protein group has been carried out to H37Rv bacterial strain using high covering protein technique.It is based on Tuberculosis (20160307) database has carried out annotation encoding gene to its genome using 3 engine of pFind and has verified. In order to find new protein-coding region, we are based on protein gene omics technology, are sent out in NCBI with pAnno software H37Rv 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 It is S-FDR, T-FDR I and T-FDR respectively kind to peptide fragment has been annotated and new peptide fragment separately estimates the filter method of classification FDR II。

Analyzed through data, we identify 3238 H37Rv altogether and have annotated gene, 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, we obtain new peptide fragment after 3 kinds of FDR≤1 are filtered. In order to further ensure that new peptide fragment quality, 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 check these higher peptide fragments of 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 Rv0007A (+| 10966-11121 |) gene

After the verifying of excessively high covering protein group, it has been found that some doubtful new leakages annotate peptide fragment, can to above-mentioned height Doubtful new peptide fragment progress peptide fragment synthesis verifying is believed to obtain, according to new peptide fragment original spectrum and peptide fragment synthesis spectrum similarity >=0.9 conduct of marking Similarity threshold has 1 peptide fragment by verifying, corresponds to new open reading frame (Open Reading after marking screening Frame, ORF), i.e., the potential leakage of current H37Rv bacterial strain annotates gene.

Wherein, it has been found that new leakage annotation gene Rv0007A (+| 10966-11121 |), compare through BLASTP, phase It has been annotated in M.tuberculosis 49-02 bacterial strain with sequence as Hypothetical protein, similar sequences exist Annotation is in the bacterial strains such as M.bovis BCG str.Korea 1168P, M.tuberculosis CAS/NITR204 Hypothetical protein, therefore the gene belongs to leakage annotation gene in H37Rv.We detect peptide fragment FALPLAAIAVAAIVVR (SEQ ID NO.6), and correspond to new gene Rv0007A (+| 10966-11121 |), such as Fig. 1 institute Show, spectrogram quality is fine, and b/y ion continuous coupling, miscellaneous peak signal is lower, as a result very credible.

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

The high energy collision MS that we generate section of synthesized peptide₂It is verified, level-one parent ion and second level daughter ion are equal Meet theoretical value, the peptide section sequence for showing that we synthesize is correct；On this basis, we have checked according to large-scale protein by hand The MS of the section of synthesized peptide for the 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 value for showing the acquisition of daughter ion similitude is 0.96, 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 the open reading frame comprising above-mentioned new leakage annotation peptide fragment (ORF) DNA sequence dna, as shown in SEQ ID NO.2.

TAGCTCAGGCGGTTAGAGCGCTTCGCTGATAACGAAGAGGTCGGAGGTTCGAGTCCTCCTAGGCCCAC GACCATGTGCCCGTCACGACGTTCGGTGAGGTTCGCATTGCCACTGGCCGCGATCGCTGTGGCGGCCATCGTCGTG CGGTTCCGACGCGGAGCCGATGTCTGGCATGTGGCCGGCGATCCACCTCCTGATCACATAACCGGTGACGAAGAGG GGCCTTAG(SEQ ID NO.2)

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

Further translation verifying, finds true gene order (SEQ ID NO.1) from above-mentioned open reading frame DNA (SEQ ID NO.2) inATGStarting, total 156bp encodes 43 amino acid, theoretical molecular weight 5.48kDa, as Rv0007A (+| 10966-11121 |) gene.

ATGTGCCCGTCACGACGTTCGGTGAGGTTCGCATTGCCACTGGCCGCGATCGCTGTGGCGGCCATCGT CGTGCGGTTCCGACGCGGAGCCGATGTCTGGCATGTGGCCGGCGATCCACCTCCTGATCACATAACCGGTGACGAA GAGGGGCCTTAG(SEQ ID NO.1)

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

MCPSRRSVRFALPLAAIAVAAIVVRFRRGADVWHVAGDPPPDHITGDEEGP(SEQ ID NO.3)

NCBI-BLASTP analysis is carried out to the amino acid sequence of theory gene encoding production shown in the SEQ ID NO.3, Identical sequence in M.tuberculosis 49-02 bacterial strain, similar sequences M.bovis BCG str.Korea 1168P, Annotation is Hypothetical protein in M.tuberculosis CAS/NITR204 bacterial strain.(see Fig. 5).Sufficiently show me Rv0007A (+| 10966-11121 |) gene product for detecting be missed annotation in H37Rv bacterial strain database.

The DNA sequence dna of the Rv0007A (+| 10966-11121 |) gene is compared genome Local BLAST point by us Analysis, as shown in Figure 4, the results showed that Rv0007A (+| 10966-11121 |) gene order belongs to MTBC family specificity gene, There is no the higher sequence of homology in other species, it is meant that Rv0007A that we have found in H37Rv bacterial strain (+| 10966- 11121 |) gene order has preferable sequence-specific, MTBC and it can will belong to interior other mycobacterias and other respiratory tracts Bacterial infection distinguishes.

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

(1) design primer:

Based on the CDS sequence of the Rv0007A as shown in SEQ ID NO.1 (+| 10966-11121 |) gene, use Oligo7.0 devises PCR primer, and primer sequence is as follows:

F:5'-TGTGCCCGTCACGACGTTCGGTGAG-3'(SEQ ID NO.4)；

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

Above-mentioned primer is as follows with the positional relationship of Rv0007A (+| 10966-11121 |) gene, wherein primer pair Position subscript list is answered to cross.

ATGTGCCCGTCACGACGTTCGGTGAGGTTCGCATTGCCACTGGCCGCGATCGCTGTGGCGGCCATCGT CGTGCGGTTCCGACGCGGAGCCGATGTCTGGCATGTGGCCGGCGATCCACCTCCTGATCACATAACCGGTGACGAA GAGGGGCCTTAG(SEQ ID NO.1)

(2) total DNA of the strain to be tested including M.tuberculosis H37Rv, 40 plants of Mycobacterium marks are extracted 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 the sequencing of strain 16S rna gene, comparison and NCBI sequence 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) reaction, and above-mentioned F/R primer used is expanded.

PCR system (25 μ L) is dd H₂O (9.5 μ L), 2XTaq PCR MasterMix (TIANGEN, 12.5 μ L), primer F (10 μM, 1 μ L), primer R (10 μM, 1 μ L), DNA profiling (1 μ L)；

Amplification program: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of 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 liquid.As a result such as Fig. 6 institute Show, amplified band occur at 155bp in MTBC and positive controls, and amplification is consistent with expection, and specificity is 100%.

(5) in order to further verify amplification DNA sequence, we to extension increasing sequence carried out sequencing and and leakage annotation sequence Column compare, as shown in fig. 7, result is consistent completely with expection, sequence is correct, this further demonstrates new leakage annotation gene In the presence of.This shows that the method that the compound group's identification of MTBC is carried out based on Rv0007A (+| 10966-11121 |) gene is true and reliable.

SEQUENCE LISTING

<110>Beijing Proteome Research Center

<120>Mycobacterium tuberculosis H37Rv encoding gene and its application

<130> BJ1936-18P121913

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 156

<212> DNA

<213> Artificial

<220>

<223>Mycobacterium tuberculosis H37Rv encoding gene Rv0007A (+| 10966-11121 |)

<400> 1

atgtgcccgt cacgacgttc ggtgaggttc gcattgccac tggccgcgat cgctgtggcg 60

gccatcgtcg tgcggttccg acgcggagcc gatgtctggc atgtggccgg cgatccacct 120

cctgatcaca taaccggtga cgaagagggg ccttag 156

<210> 2

<211> 228

<212> DNA

<213> Artificial

<220>

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

<400> 2

tagctcaggc ggttagagcg cttcgctgat aacgaagagg tcggaggttc gagtcctcct 60

aggcccacga ccatgtgccc gtcacgacgt tcggtgaggt tcgcattgcc actggccgcg 120

atcgctgtgg cggccatcgt cgtgcggttc cgacgcggag ccgatgtctg gcatgtggcc 180

ggcgatccac ctcctgatca cataaccggt gacgaagagg ggccttag 228

<210> 3

<211> 51

<212> PRT

<213> Artificial

<220>

<223>Rv0007A (+| 10966-11121 |) gene theory coded product amino acid sequence

<400> 3

Met Cys Pro Ser Arg Arg Ser Val Arg Phe Ala Leu Pro Leu Ala Ala

1 5 10 15

Ile Ala Val Ala Ala Ile Val Val Arg Phe Arg Arg Gly Ala Asp Val

20 25 30

Trp His Val Ala Gly Asp Pro Pro Pro Asp His Ile Thr Gly Asp Glu

35 40 45

Glu Gly Pro

50

<210> 4

<211> 25

<212> DNA

<213> Artificial

<220>

<223>F primer sequence

<400> 4

tgtgcccgtc acgacgttcg gtgag 25

<210> 5

<211> 25

<212> DNA

<213> Artificial

<220>

<223>R primer sequence

<400> 5

ctaaggcccc tcttcgtcac cggtt 25

<210> 6

<211> 16

<212> PRT

<213> Artificial

<220>

<223>leakage annotation peptide fragment

<400> 6

Phe Ala Leu Pro Leu Ala Ala Ile Ala Val Ala Ala Ile Val Val Arg

1 5 10 15

Claims (10)

1. a kind of Mycobacterium tuberculosis H37Rv encoding gene Rv0007A (+| 10966-11121 |), the nucleosides of the encoding gene Acid sequence is as shown in SEQ ID NO.1.

2. Mycobacterium tuberculosis H37Rv encoding gene Rv0007A described in claim 1 (+| 10966-11121 |), feature It is gene coding amino acid as shown in SEQ ID NO.3 sequence.

3. a kind of bar code molecular labeling is used as and detects and/or identify mycobacterium tuberculosis complex, it includes examine as standard The Mycobacterium tuberculosis H37Rv encoding gene Rv0007A described in claim 1 (+| 10966-11121 |) of cls gene.

4. a species-specific PCR primers, for expanding Mycobacterium tuberculosis H37Rv encoding gene described in claim 1 Rv0007A(+|10966-11121|)。

5. PCR primer as claimed in claim 4, which is characterized in that the sequence of the primer are as follows:

F:5'-TGTGCCCGTCACGACGTTCGGTGAG-3'；

R:5’-CTAAGGCCCCTCTTCGTCACCGGTT-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) amplimer is added as template in the DNA obtained using step (1), carries out polymerase chain reaction；

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

(4) by the result of step (3) and it is described in claim 1 as standard detection gene Rv0007A (+| 10966- 11121 |) be compared, according in its homologous sex determination sample to be tested whether there is mycobacterium tuberculosis complex.

7. detection method as claimed in claim 6, wherein amplimer sequence described in step (2) are as follows:

F:5'-TGTGCCCGTCACGACGTTCGGTGAG-3'；

R:5’-CTAAGGCCCCTCTTCGTCACCGGTT-3’。

8. detection method as claimed in claim 6, wherein if homology is greater than 99%, determining to test sample in step (4) Contain mycobacterium tuberculosis complex in product.

9. a kind of detection kit, comprising Mycobacterium tuberculosis H37Rv encoding gene Rv0007A described in claim 1 (+| 10966-11121 |) and/or Specific PCR primers as claimed in claim 4.

10. Mycobacterium tuberculosis H37Rv encoding gene Rv0007A described in claim 1 (+| 10966-11121 |) in tuberculosis Application in epidemiological survey and/or clinical tuberculosis patient quick diagnosis and identification.