CN110408630A - Mycobacterium tuberculosis H37Rv encoding gene and its application - Google Patents
Mycobacterium tuberculosis H37Rv encoding gene and its application Download PDFInfo
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Abstract
The present invention relates to a kind of Mycobacterium tuberculosis H37Rv encoding gene and its applications, more particularly to new leakage annotation encoding gene Rv0229A (- | 274710-274904 |), its standard gene that can be used as mycobacterium tuberculosis complex Molecular Identification, Molecular Identification and clinical detection for mycobacterium tuberculosis complex.
Description
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 Rv0229A (- | 274710-274904 |), 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 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. Rv0229A (- | 274710-274904 |), after NCBI-BLASTP, any sequence is arrived without comparing in database, is belonged to
In Unknown Function albumen.It is studied through comparative genomics and finds that the gene order can be by mycobacterium tuberculosis complex (MTBC) bacterium
Strain is distinguished with other kinds of Mycobacterium.
Specifically, design can Rv0229A (- | 274710-274904 |) gene to MTBC realize specificity amplification primer,
Primer as proposed by the invention, primer sequence are as follows:
F:5'-GGCATTACCTCCACATCCAC-3'；
R:5’-CCTCAGCCAACGGTTCCA-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'-GGCATTACCTCCACATCCAC-3'(SEQ ID NO.4)；
R:5’-CCTCAGCCAACGGTTCCA-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 Rv0229A (- | 274710-274904 |) are compared, if
Homology 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 Rv0229A of H37Rv (- | 274710-274904 |) carries out Homology search and comparison, obtains
Similitude between sequence 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 distinguish MTBC family and non-tuberculous mycobacteria, respiratory tract common disease
Opportunistic 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 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 Rv0229A in product (- | 274710-274904 |) 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；
A: the first peptide fragment B, C: Article 2 peptide fragment D: Article 3 peptide fragment
Fig. 2: section of synthesized peptide mass spectrogram and former identification peptide fragment mass spectrogram comparison；
A: the first peptide fragment B: Article 2 peptide fragment C: Article 3 peptide fragment
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: Rv0229A (- | 274710-274904 |) standard gene sequence homology compares；
Protein sequence BLASTP result corresponding to Fig. 5: H37Rv bacterial strain Rv0229A (- | 274710-274904 |) gene；
Fig. 6: Rv0229A (- | 274710-274904 |) specific primer PCR amplified production agarose gel electrophoresis results；
Wherein, each Lane Sample specifying information is shown in Table 1.
Fig. 7: Rv0229A (- | 274710-274904 |) 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 Rv0229A (- | 274710-274904 |) 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 3 peptide fragments 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 Rv0229A (- | 274710-274904 |), compare through BLASTP,
It is Hypothetical protein that identical sequence annotates in ncbi database, and similar sequences are in M.tuberculosis
It has been noted as Antitoxin in 88 bacterial strain of Beijing/MYC004 and M.tuberculosis Bir, may have been caused with MTB bacterium
The relevant property of characteristic of disease, therefore the gene belongs to leakage annotation gene in H37Rv.We detect peptide fragment
TELGTTTIKDTVNAALR(SEQ ID NO.6)、VAAALDTLAAAPPEDR(SEQ ID NO.7)、HLVDIDEQALNMAR
(SEQ ID NO.8), and correspond to new gene Rv0229A (- | 274710-274904 |), as shown in Figure 1, spectrogram quality is fine,
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
Unanimously, TELGTTTIKDTVNAALR similitude cosin value is 0.98, VAAALDTLAAAPPEDR daughter ion cosin value is
0.94, HLVDIDEQALNMAR daughter ion cosin value is 0.99, 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.
TGATATATACTCCGATTCATGGCGAAACATCTCGTCGACATCGACGAGCAGGCTTTAAACATGGCTCGTACAGAATT
GGGCACGACGACGATCAAAGACACCGTCAACGCGGCCCTGCGGCAAGCCACGTCTCAGCGAGTTCAACGCGTCGCCG
CCGCTCTCGACACGCTGGCCGCCGCACCGCCAGAGGACCGCGCCGAAGCATGGCGCTGA(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 195bp encodes 64 amino acid, theoretical molecular weight 6.97kDa, as Rv0229A (- |
274710-274904 |) gene.
ATGGCGAAACATCTCGTCGACATCGACGAGCAGGCTTTAAACATGGCTCGTACAGAATTGGGCACGACGACGATCAA
AGACACCGTCAACGCGGCCCTGCGGCAAGCCACGTCTCAGCGAGTTCAACGCGTCGCCGCCGCTCTCGACACGCTGG
CCGCCGCACCGCCAGAGGACCGCGCCGAAGCATGGCGCTGA(SEQ ID NO.1)
The gene theory coded product amino acid sequence is as shown in SEQ ID NO.3:
MAKHLVDIDEQALNMARTELGTTTIKDTVNAALRQATSQRVQRVAAALDTLAAAPPEDRAEAWR(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,
Same or similar sequence is in RefSeq database, M.tuberculosis Beijing/MYC004 and M.tuberculosis
Antitoxin albumen is noted as in 88 bacterial strain of Bir, neighboring gene Rv0229c is Toxin albumen, which annotates gene
A pair of of toxin-antitoxin system (Toxin-antitoxin system) is just constituted with Rv0229c, may be caused a disease with MTB bacterium
Property related (see Fig. 5).Sufficiently show Rv0229A that we detect (- | 274710-274904 |) gene product in H37Rv bacterium
Annotation is missed in strain database.
The DNA sequence dna of the Rv0229A (- | 274710-274904 |) gene is compared genome Local BLAST by us
Analysis, as shown in Figure 5, the results showed that Rv0229A (- | 274710-274904 |) gene order belongs to MTBC family specificity base
Cause does not have the higher sequence of homology in other species, it is meant that Rv0229A that we have found in H37Rv bacterial strain (- |
274710-274904 |) gene order have preferable sequence-specific, can by MTBC and belong to interior other mycobacterias and its
Its Bacteria infecting respiratory 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 Rv0229A as shown in SEQ ID NO.1 (- | 274710-274904 |) gene, use
Oligo7.0 devises PCR primer, and primer sequence is as follows:
F:5'-GGCATTACCTCCACATCCAC-3'(SEQ ID NO.4)；
R:5’-CCTCAGCCAACGGTTCCA-3’(SEQ ID NO.5)
Above-mentioned primer is as follows with the positional relationship of Rv0229A (- | 274710-274904 |) gene, wherein primer
The scribing line of corresponding position subscript list, gray background are the complete ORF sequence of leakage annotation gene Rv0229A (- | 274710-274904 |).
GGCATTACCTCCACATCCACAACGACGTCATCCCCGCACTGAAGCAGCACGGCGTCACCGACGAGCAGCTGCACACC
ATGCTCGTCGACAACCCGCGCCGCATCTTCGAGCGGCAGGGCGGCTATCAGTGAGACAGCCGCGCCGGGCGAATGCC
ATGGGCTTGGCATTGTGCATATATATCGGCTCCTTATTGATATATACTCCGATTCATGGCGAAACATCTCGTCGACA
TCGACGAGCAGGCTTTAAACATGGCTCGTACAGAATTGGGCACGACGACGATCAAAGACACCGTCAACGCGGCCCTG
CGGCAAGCCACGTCTCAGCGAGTTCAACGCGTCGCCGCCGCTCTCGACACGCTGGCCGCCGCACCGCCAGAGGACCG
CGCCGAAGCATGGCGCTGAAATATCTTCTCGACACCAGCGTGATCAAAAGGCTCAGCCGGCCCGCCGTGCGGCGGGC
GGTGGAACCGTTGGCTGAGG(SEQ ID NO.9)
(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 482bp in MTBC and positive controls, and amplification is consistent with expection, and specificity is
96.7%.
(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 Rv0229A (- | 274710-274904 |) gene is true and reliable.
SEQUENCE LISTING
<110>Beijing Proteome Research Center
<120>Mycobacterium tuberculosis H37Rv encoding gene and its application
<130> BJ1936-18P121914
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 195
<212> DNA
<213> Artificial
<220>
<223>Mycobacterium tuberculosis H37Rv encoding gene Rv0229A (- | 274710-274904 |)
<400> 1
atggcgaaac atctcgtcga catcgacgag caggctttaa acatggctcg tacagaattg 60
ggcacgacga cgatcaaaga caccgtcaac gcggccctgc ggcaagccac gtctcagcga 120
gttcaacgcg tcgccgccgc tctcgacacg ctggccgccg caccgccaga ggaccgcgcc 180
gaagcatggc gctga 195
<210> 2
<211> 213
<212> DNA
<213> Artificial
<220>
<223>the open reading frame DNA sequence dna comprising leakage annotation peptide fragment
<400> 2
tgatatatac tccgattcat ggcgaaacat ctcgtcgaca tcgacgagca ggctttaaac 60
atggctcgta cagaattggg cacgacgacg atcaaagaca ccgtcaacgc ggccctgcgg 120
caagccacgt ctcagcgagt tcaacgcgtc gccgccgctc tcgacacgct ggccgccgca 180
ccgccagagg accgcgccga agcatggcgc tga 213
<210> 3
<211> 64
<212> PRT
<213> Artificial
<220>
<223>Rv0229A (- | 274710-274904 |) gene theory coded product amino acid sequence
<400> 3
Met Ala Lys His Leu Val Asp Ile Asp Glu Gln Ala Leu Asn Met Ala
1 5 10 15
Arg Thr Glu Leu Gly Thr Thr Thr Ile Lys Asp Thr Val Asn Ala Ala
20 25 30
Leu Arg Gln Ala Thr Ser Gln Arg Val Gln Arg Val Ala Ala Ala Leu
35 40 45
Asp Thr Leu Ala Ala Ala Pro Pro Glu Asp Arg Ala Glu Ala Trp Arg
50 55 60
<210> 4
<211> 20
<212> DNA
<213> Artificial
<220>
<223>F primer sequence
<400> 4
ggcattacct ccacatccac 20
<210> 5
<211> 18
<212> DNA
<213> Artificial
<220>
<223>R primer sequence
<400> 5
cctcagccaa cggttcca 18
<210> 6
<211> 17
<212> PRT
<213> Artificial
<220>
<223>first leakages annotate peptide fragment
<400> 6
Thr Glu Leu Gly Thr Thr Thr Ile Lys Asp Thr Val Asn Ala Ala Leu
1 5 10 15
Arg
<210> 7
<211> 16
<212> PRT
<213> Artificial
<220>
<223>Article 2 leakage annotation peptide fragment
<400> 7
Val Ala Ala Ala Leu Asp Thr Leu Ala Ala Ala Pro Pro Glu Asp Arg
1 5 10 15
<210> 8
<211> 14
<212> PRT
<213> Artificial
<220>
<223>Article 3 leakage annotation peptide fragment
<400> 8
His Leu Val Asp Ile Asp Glu Gln Ala Leu Asn Met Ala Arg
1 5 10
<210> 9
<211> 482
<212> DNA
<213> Artificial
<220>
<223>integration sequence of upstream and downstream primer and Rv0229A (- | 274710-274904 |) gene is demonstrated
<400> 9
ggcattacct ccacatccac aacgacgtca tccccgcact gaagcagcac ggcgtcaccg 60
acgagcagct gcacaccatg ctcgtcgaca acccgcgccg catcttcgag cggcagggcg 120
gctatcagtg agacagccgc gccgggcgaa tgccatgggc ttggcattgt gcatatatat 180
cggctcctta ttgatatata ctccgattca tggcgaaaca tctcgtcgac atcgacgagc 240
aggctttaaa catggctcgt acagaattgg gcacgacgac gatcaaagac accgtcaacg 300
cggccctgcg gcaagccacg tctcagcgag ttcaacgcgt cgccgccgct ctcgacacgc 360
tggccgccgc accgccagag gaccgcgccg aagcatggcg ctgaaatatc ttctcgacac 420
cagcgtgatc aaaaggctca gccggcccgc cgtgcggcgg gcggtggaac cgttggctga 480
gg 482
Claims (10)
1. a kind of Mycobacterium tuberculosis H37Rv encoding gene Rv0229A (- | 274710-274904 |), the core of the encoding gene
Nucleotide sequence is as shown in SEQ ID NO.1.
2. Mycobacterium tuberculosis H37Rv encoding gene Rv0229A described in claim 1 (- | 274710-274904 |), it is special
Sign 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 Rv0229A described in claim 1 (- | 274710-274904 |) of cls gene.
4. a species-specific PCR primers, for expanding Mycobacterium tuberculosis H37Rv encoding gene described in claim 1
Rv0229A(-|274710-274904|)。
5. PCR primer as claimed in claim 4, which is characterized in that the sequence of the primer are as follows:
F:5'-GGCATTACCTCCACATCCAC-3'；
R:5’-CCTCAGCCAACGGTTCCA-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 Rv0229A (- | 274710-
274904 |) 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'-GGCATTACCTCCACATCCAC-3'；
R:5’-CCTCAGCCAACGGTTCCA-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 Rv0229A described in claim 1 (- |
274710-274904 |) and/or Specific PCR primers as claimed in claim 4.
10. Mycobacterium tuberculosis H37Rv encoding gene Rv0229A described in claim 1 (- | 274710-274904 |) it is tying
Application in core epidemiological survey and/or clinical tuberculosis patient quick diagnosis and identification.
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| CN113373162A (en) * | 2020-03-09 | 2021-09-10 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv novel gene Rv0229A and encoding protein and application thereof |
| CN113403327A (en) * | 2020-03-17 | 2021-09-17 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv new gene Rv2706 and coding protein and application thereof |
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| CN113373162A (en) * | 2020-03-09 | 2021-09-10 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv novel gene Rv0229A and encoding protein and application thereof |
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