CN108165563A - Mycobacterium tuberculosis H37Rv encoding gene and its application - Google Patents
Mycobacterium tuberculosis H37Rv encoding gene and its application Download PDFInfo
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- 241001646725 Mycobacterium tuberculosis H37Rv Species 0.000 title claims abstract description 15
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- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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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
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, complex steps, specifically
The defects of property and sensitivity is not high.H37Rv full-length genomes are annotated again further to improve, find to omit annotation in H37Rv
Gene, it is ensured that H37Rv full-length genomes omit annotation gene and its application technology in MTBC Molecular Identifications is effectively protected,
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 Rv3202B (+| 3580526-3580684 |), it can be used as mycobacterium tuberculosis complex bar code point
Son 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. Rv3202B (+| 3580526-3580684 |), after NCBI-BLASTP, with mycobacterium tuberculosis family species
Lipase LipV protein similarities are higher, as M.tuberculosis NRITLD44, M.africanum MAL010071,
M.canetti RefSeq (WP_041180143.1) similitude is 88%, and 74% is below with the similitude of other bacterial strains.Through
Comparative genomics research finds that the gene order can be by mycobacterium tuberculosis complex (MTBC) bacterial strain and Mycobacterium
Other kinds distinguish.
Specifically, design can realize that specific amplification draws to the Rv3202B of MTBC (+| 3580526-3580684 |) gene
Object, primer as proposed by the invention, primer sequence are：
F:5’-TCATCGACCTTCACGTACA-3’；
R:5’-AAAGTGATGGGCTAACCG-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’-TCATCGACCTTCACGTACA-3’(SEQ ID NO.4)；
R:5’-AAAGTGATGGGCTAACCG-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 Rv3202B (+| 3580526-3580684 |) are compared, such as
Fruit 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 Rv3202B of H37Rv (+| 3580526-3580684 |) 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 Rv3202B in product (+| 3580526-3580684 |) gene, for example, containing amplimer, for the DNA of PCR reactions
Polymerase and its buffer solution, endonuclease reaction and/or reagent needed for sequencing reaction etc. it is one or more.Those skilled in the art are
Know, more than component is only illustrative, for example, above-mentioned Specific PCR primers may be used in the primer, described is used for PCR
The archaeal dna polymerase of reaction can be used for the enzyme of PCR amplification.The detection of the encoding gene of the present invention can also with it is integrated for example
The mode of genetic chip provides.
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：Rv3202B (+| 3580526-3580684 |) standard gene sequence homology compares；
Fig. 5：Protein sequence BLASTP knots corresponding to H37Rv bacterial strains Rv3202B (+| 3580526-3580684 |) gene
Fruit；
Fig. 6：Rv3202B (+| 3580526-3580684 |) specific primer PCR amplified production agarose gel electrophoresis result；
Wherein, each Lane Sample specifying information is shown in Table 1.
Fig. 7：Rv3202B (+| 3580526-3580684 |) 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 Rv3202B (+| 3580526-3580684 |) 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 Rv3202B (+| 3580526-3580684 |), compare through BLASTP,
It is higher with the Lipase LipV protein similarities of mycobacterium tuberculosis family species, as M.tuberculosis NRITLD44,
M.africanum MAL010071, M.canetti RefSeq (WP_041180143.1) similitude are 88%, with other bacterial strains
Similitude be below 74%.We detect peptide fragment VLTIHGVTEHGR (SEQ ID NO.6), and corresponding to new gene
Rv3202B (+| 3580526-3580684 |), as shown in Figure 1, spectrogram quality is fine, b/y ion continuous couplings, miscellaneous peak signal is very
It is low, it is 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 0.97, 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.
TAGCAGGCGTCGCCGGGCTGGCATCATCGACGCGTGATCATCGACCTTCACGTACAGCGCTACGGCCCGTCAGGGCC
CGCGCGGGTGCTGACCATCCACGGAGTGACCGAGCACGGGCGCATCTGGCACCGGTTAGCCCATCACTTTGCCCGAA
ATCCCCATCGCCGCACCCGATCTGCTGGGCCACGGTAG(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) inGTGStart, common 159bp, 52 amino acid of coding, theoretical molecular weight 5.99kDa, as Rv3202B (+|
3580526-3580684 |) gene.
GTGATCATCGACCTTCACGTACAGCGCTACGGCCCGTCAGGGCCCGCGCGGGTGCTGACCATCCACGGAGTGACCGA
GCACGGGCGCATCTGGCACCGGTTAGCCCATCACTTTGCCCGAAATCCCCATCGCCGCACCCGATCTGCTGGGCCAC
GGTAG(SEQ ID NO.1)
The gene theory coded product amino acid sequence is as shown in SEQ ID NO.3：
VIIDLHVQRYGPSGPARVLTIHGVTEHGRIWHRLAHHFARNPHRRTRSAGPR(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
The Lipase LipV protein similarities of mycobacterium tuberculosis family species are higher, as M.tuberculosis NRITLD44,
M.africanum MAL010071, M.canetti RefSeq (WP_041180143.1) similitude are 88%, with other bacterial strains
Similitude be below 74%.(see Fig. 4).Show Rv3202B that we detect (+| 3580526-3580684 |) gene production
Object is missed annotation in H37Rv bacterial strain databases.
The DNA sequence dna of the Rv3202B (+| 3580526-3580684 |) gene is compared genome local by us
BLAST is analyzed, as shown in Figure 5, the results showed that Rv3202B (+| 3580526-3580684 |) gene order belongs to MTBC families spy
Specific gene, the sequence for not having homology higher in other species, this shows what we had found in H37Rv bacterial strains
Rv3202B (+| 3580526-3580684 |) gene order has preferable sequence-specific, can be interior other with belonging to by MTBC
Mycobacteria 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 Rv3202B (+| 3580526-3580684 |) gene as shown in SEQ ID NO.1, use
Oligo7.0 devises PCR primer, and primer sequence is as follows：
F:5’-TCATCGACCTTCACGTACA-3’(SEQ ID NO.4)；
R:5’-AAAGTGATGGGCTAACCG-3’(SEQ ID NO.5)
Above-mentioned primer is as follows with the position relationship of Rv3202B (+| 3580526-3580684 |) gene, wherein drawing
Object corresponding position subscript list is crossed.
GTGATCATCGACCTTCACGTACAGCGCTACGGCCCGTCAGGGCCCGCGCGGGTGCTGACCATCCACGGAGTGACCGA
GCACGGGCGCATCTGGCACCGGTTAGCCCATCACTTTGCCCGAAATCCCCATCGCCGCACCCGATCTGCTGGGCCAC
GGTAG(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 the sequencing of strain 16S rna genes, comparison and NCBI sequences and have submitted work,
Strain to be tested is as shown in table 1：
The related strain serial number 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 are at 110bp) there is amplified band, and amplification is consistent with expection, specificity is 96.7%.
(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 Rv3202B (+| 3580526-3580684 |) gene is true
Reliably.
SEQUENCE LISTING
<110>Beijing Proteome Research Center
<120>Mycobacterium tuberculosis H37Rv encoding gene and its application
<130> BJ1936-17P121795
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 159
<212> DNA
<213> Artificial
<220>
<223>Mycobacterium tuberculosis H37Rv encoding gene Rv3202B (+| 3580526-3580684 |)
<400> 1
gtgatcatcg accttcacgt acagcgctac ggcccgtcag ggcccgcgcg ggtgctgacc 60
atccacggag tgaccgagca cgggcgcatc tggcaccggt tagcccatca ctttgcccga 120
aatccccatc gccgcacccg atctgctggg ccacggtag 159
<210> 2
<211> 192
<212> DNA
<213> Artificial
<220>
<223>Include the open reading frame DNA sequence dna of leakage annotation peptide fragment
<400> 2
tagcaggcgt cgccgggctg gcatcatcga cgcgtgatca tcgaccttca cgtacagcgc 60
tacggcccgt cagggcccgc gcgggtgctg accatccacg gagtgaccga gcacgggcgc 120
atctggcacc ggttagccca tcactttgcc cgaaatcccc atcgccgcac ccgatctgct 180
gggccacggt ag 192
<210> 3
<211> 52
<212> PRT
<213> Artificial
<220>
<223>Rv3202B (+| 3580526-3580684 |) gene theory coded product amino acid sequence
<400> 3
Val Ile Ile Asp Leu His Val Gln Arg Tyr Gly Pro Ser Gly Pro Ala
1 5 10 15
Arg Val Leu Thr Ile His Gly Val Thr Glu His Gly Arg Ile Trp His
20 25 30
Arg Leu Ala His His Phe Ala Arg Asn Pro His Arg Arg Thr Arg Ser
35 40 45
Ala Gly Pro Arg
50
<210> 4
<211> 19
<212> DNA
<213> Artificial
<220>
<223>F primer sequences
<400> 4
tcatcgacct tcacgtaca 19
<210> 5
<211> 18
<212> DNA
<213> Artificial
<220>
<223>R primer sequences
<400> 5
aaagtgatgg gctaaccg 18
<210> 6
<211> 12
<212> PRT
<213> Artificial
<220>
<223>Leakage annotation peptide fragment
<400> 6
Val Leu Thr Ile His Gly Val Thr Glu His Gly Arg
1 5 10
Claims (10)
1. a kind of Mycobacterium tuberculosis H37Rv encoding gene, the nucleotide sequence of the encoding gene is as shown in SEQID NO.1.
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’-TCATCGACCTTCACGTACA-3’；
R:5’-AAAGTGATGGGCTAACCG-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；
(3) DNA product obtained to step (2) amplification carries out gel electrophoresis analysis or is sequenced；
(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’-TCATCGACCTTCACGTACA-3’；
R:5’-AAAGTGATGGGCTAACCG-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.
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