CN108004253A - Mycobacterium tuberculosis H37Rv encoding gene and its application - Google Patents
Mycobacterium tuberculosis H37Rv encoding gene and its application Download PDFInfo
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- CN108004253A CN108004253A CN201711249300.3A CN201711249300A CN108004253A CN 108004253 A CN108004253 A CN 108004253A CN 201711249300 A CN201711249300 A CN 201711249300A CN 108004253 A CN108004253 A CN 108004253A
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Abstract
The present invention relates to a kind of Mycobacterium tuberculosis H37Rv encoding gene, it can be used as the standard gene of mycobacterium tuberculosis complex Molecular Identification, Molecular Identification and clinical detection for mycobacterium tuberculosis complex.
Description
Technical field
The present invention relates to genetic test field, 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, 8,000,000 new cases generation there are about every year, at least 3,000,000 people die of the disease.The clinical bacterium of MTB
The culture of strain hardly possible, slow-growing and other mycobacteria energy cross-infections, tuberculosis and the difficult differentiation of other respiratory tract infection symptoms etc.
Feature, great difficulty is brought to clinical quick diagnosis and treatment.Therefore establish quick, accurate, special, sensitive, cheap knot
Core disease detection method, is effective treatment, the prerequisite of control tuberculosis sprawling, and clinical labororatory's mycobacteria detection faces
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 mycobacteria 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
Survey (IS6110, restriction fragment length polymorphism analysis, more site 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 cycle 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.
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 renewal, 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 cycle 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 H37Rv new genes fast accurate mirror method for distinguishing in MTBC monoids.
The content of the invention
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 Rv0609B (- | 704475-704672 |), it can be used as mycobacterium tuberculosis complex bar code molecule
Mark, for detecting mycobacterium tuberculosis complex, its 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, comparison protein genomics investigative technique is passed through, 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 together
Come.The gene is the omission annotation base of a mycobacterium tuberculosis (Mycobacterium tuberculosis H37Rv)
Cause, i.e. Rv0609B (- | 704475-704672 |), after NCBI-BLASTP, with M.bovis RefSeq (WP_
003403191.1) similitude with M.tuberculosis Bir189 is 98%, M.canettii CIPT, 140070017 phases
It is 94% like property, other species similarities are below 34%, and belong to Unknown Function albumen.Study and send out through comparative genomics
Now other kinds of mycobacterium tuberculosis complex (MTBC) bacterial strain and Mycobacterium can be distinguished by the gene order.
Specifically, design can realize specificity amplification primer to the Rv0609B of MTBC (- | 704475-704672 |) gene,
Primer as proposed by the invention, primer sequence are:
F:5’-GACCGACGAGAAGTGCGT-3’;
R:5’-GGCACGGCTTTCGAGATA-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’-GACCGACGAGAAGTGCGT-3’(SEQ ID NO.4);
R:5’-GGCACGGCTTTCGAGATA-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 Rv0609B (- | 704475-704672 |) are compared, if
Homology is more than 99%, judges that 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 band, then can further sequence verification, will survey
The standard sequence that sequence obtains series and the Rv0609B of H37Rv (- | 704475-704672 |) carries out Homology search and comparison, obtains
Similitude between sequence, if sequence homology is more than 99%, you can judge that bacterial strain may be MTBC;According to identification bacterial strain to be identified
DNA bar code sequence and standard sequence cluster situation distinguish MTBC families 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
Examine.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 use tuberculosis and other respiratory tract patient sputum, 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 medicine or biological products.For example, after using PCR amplification, sample is directly detected
The reagent of Rv0609B in product (- | 704475-704672 |) gene, for example, containing amplimer, dNTP, for PCR reactions
The one or more of archaeal dna polymerase and its buffer solution, endonuclease reaction and/or reagent needed for sequencing reaction etc..People in the art
Member is it is known that 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 reactions can be used for the enzyme of PCR amplification.The detection of the encoding gene of the present invention can also be with integrated
Such as the mode of genetic chip provides.
Beneficial 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 together, 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, have the characteristics that general, easy amplification, easily compare, can
To identify to come from the close other mycobacterias of affiliation or other respiratory tract infection germs by the monoid exactly, it is
Tuberculosis epidemiology survey and clinical tuberculosis patient quick diagnosis, differentiate the strong technological means of offer and research tool.
Brief description of the drawings
Fig. 1:Support the peptide spectrum matching evidence of newly encoded gene found;
Fig. 2:Section of synthesized peptide mass spectrogram is contrasted with former identification peptide fragment mass spectrogram;
Fig. 3:Peptide fragment is located the protein sequence corresponding diagram of region ORF codings;Identified for proteomics underscore part
And it is synthesized the peptide fragment of peptide fragment verification;
Fig. 4:Rv0609B (- | 704475-704672 |) standard gene sequence homology compares;
Fig. 5:Protein sequence BLASTP results corresponding to H37Rv bacterial strains Rv0609B (- | 704475-704672 |) gene;
Fig. 6:Rv0609B (- | 704475-704672 |) specific primer PCR amplified production agarose gel electrophoresis result;
Wherein, each Lane Sample specifying information is shown in Table 1;
Fig. 7:Rv0609B (- | 704475-704672 |) gene PCR expands sequencing result and standard sequence compares.
Embodiment
With reference to embodiment, the present invention will be further described, 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 the H37Rv protein spectrum data for reporting maximum 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 some measured peptide fragments of spectrogram matter.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 verification, it is ensured that these new peptide fragments are
H37Rv newly identifies peptide fragment.
The encoding proteins and database authentication of 1.2 couples of Rv0609B (- | 704475-704672 |) gene
After excessive covering protein group verification, it has been found that some doubtful new leakage annotation peptide fragments, 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 Rv0609B (- | 704475-704672 |), compare through BLASTP, with
The similitude of M.bovis RefSeq (WP_003403191.1) and M.tuberculosis Bir 189 is 98%,
140070017 similitudes of M.canettii CIPT are 94%, and other species similarities are below 34%, and belong to function not
Know albumen.We detect peptide fragment CGGDQLVEGAVVWNAPLR (SEQ ID NO.6), and corresponding to new gene Rv0609B (- |
704475-704672 |), as shown in Figure 1, spectrogram quality is fine, b/y ion continuous couplings, miscellaneous peak signal is very low, as a result very may be used
Letter.
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 generate using above-mentioned mass spectral analysis condition the two level spectrogram of the section of synthesized peptide.
The high energy collision MS that we produce section of synthesized peptide2Verified, 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 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, both are 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
By mistake.(Fig. 2).
After the sequence of above-mentioned leakage annotation peptide fragment is confirmed, 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.
TAGCCTGGCCAGCCGCAGAGAAGGGGAGGCGTCGTGACCGACGAGAAGTGCGTGAGATGCGGGGGCGACCAGCTCGT
CGAAGGGGCCGTCGTCTGGAACGCGCCGCTGCGTTTCAAGCGCGAAGGTGCCGGCCACTTCAATCGCGGCACCCAGG
TCAACGCCGTTGCCTGCGAGACTTGTGGGCATATCGACCTCTATCTCGAAAGCCGTGCCCGCGGCTCGACGAAGTGA
(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 real gene order (SEQ ID NO.1) from above-mentioned open reading frame DNA (SEQ
ID NO.2) inGTGStart, common 198bp, 65 amino acid of coding, its theoretical molecular 7.08kDa, as Rv0609B (- |
704475-704672 |) gene.
GTGACCGACGAGAAGTGCGTGAGATGCGGGGGCGACCAGCTCGTCGAAGGGGCCGTCGTCTGGAACGCGCCGCTGCG
TTTCAAGCGCGAAGGTGCCGGCCACTTCAATCGCGGCACCCAGGTCAACGCCGTTGCCTGCGAGACTTGTGGGCATA
TCGACCTCTATCTCGAAAGCCGTGCCCGCGGCTCGACGAAGTGA(SEQ ID NO.1)
The gene theory coded product amino acid sequence is as shown in SEQ ID NO.3:
VTDEKCVRCGGDQLVEGAVVWNAPLRFKREGAGHFNRGTQVNAVACETCGHIDLYLESRARGSTK(SEQ
ID NO.3)
NCBI-BLASTP analyses are carried out to the amino acid sequence of theoretical gene encoding production shown in the SEQ ID NO.3, with
The similitude of M.bovis RefSeq (WP_003403191.1) and M.tuberculosis Bir189 is 98%,
140070017 similitudes of M.canettii CIPT are 94%, and other species similarities are below 34%, and belong to function not
Know albumen (see Fig. 4).Show Rv0609B that we detect (- | 704475-704672 |) gene outcome in H37Rv bacterial strain numbers
According to being missed annotation in storehouse.
The DNA sequence dna of the Rv0609B (- | 704475-704672 |) gene is compared genome Local BLAST by us
Analysis, as shown in Figure 5, the results showed that Rv0609B (- | 704475-704672 |) gene order belongs to MTBC family specificity bases
Cause, does not have the higher sequence of homology in other species, this show Rv0609B that we have found in H37Rv bacterial strains (- |
704475-704672 |) gene order has preferable sequence-specific, can by MTBC and belong to together interior other mycobacterias and its
Its Bacteria infecting respiratory distinguishes.
Embodiment 2:The method for establishing the compound groups of identification of M TBC
(1) primer is designed:
Based on the CDS sequences of Rv0609B (- | 704475-704672 |) gene as shown in SEQ ID NO.1, use
Oligo7.0 devises PCR primer, and primer sequence is as follows:
F:5’-GACCGACGAGAAGTGCGT-3’(SEQ ID NO.4);
R:5’-GGCACGGCTTTCGAGATA-3’(SEQ ID NO.5)
Above-mentioned primer is as follows with the position relationship of Rv0609B (- | 704475-704672 |) gene, wherein primer
Correspondence position subscript list is rule.
GTGACCGACGAGAAGTGCGTGAGATGCGGGGGCGACCAGCTCGTCGAAGGGGCCGTCGTCTGGAACGCGCCGCTGCG
TTTCAAGCGCGAAGGTGCCGGCCACTTCAATCGCGGCACCCAGGTCAACGCCGTTGCCTGCGAGACTTGTGGGCATA
TCGACCTCTATCTCGAAAGCCGTGCCCGCGGCTCGACGAAGTGA(SEQ ID NO.1)
(2) STb gene 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 that table 1. is selected
(3) amplification of DNA fragments, carries out polymerase chain (PCR) reaction, 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, amplified band occur at 178bp in MTBC and positive controls, and actual amplification is consistent with expection, and specificity is
95%.
(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 method for showing to be carried out the compound group's identifications of MTBC based on Rv0609B (- | 704475-704672 |) gene truly may be used
Lean on.
SEQUENCE LISTING
<110>Beijing Proteome Research Center
<120>Mycobacterium tuberculosis H37Rv encoding gene and its application
<130> BJ1936-17P121792
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 198
<212> DNA
<213> Artificial
<220>
<223>Mycobacterium tuberculosis H37Rv encoding gene Rv0609B (- | 704475-704672 |)
<400> 1
gtgaccgacg agaagtgcgt gagatgcggg ggcgaccagc tcgtcgaagg ggccgtcgtc 60
tggaacgcgc cgctgcgttt caagcgcgaa ggtgccggcc acttcaatcg cggcacccag 120
gtcaacgccg ttgcctgcga gacttgtggg catatcgacc tctatctcga aagccgtgcc 180
cgcggctcga cgaagtga 198
<210> 2
<211> 231
<212> DNA
<213> Artificial
<220>
<223>Include the open reading frame DNA sequence dna of leakage annotation peptide fragment
<400> 2
tagcctggcc agccgcagag aaggggaggc gtcgtgaccg acgagaagtg cgtgagatgc 60
gggggcgacc agctcgtcga aggggccgtc gtctggaacg cgccgctgcg tttcaagcgc 120
gaaggtgccg gccacttcaa tcgcggcacc caggtcaacg ccgttgcctg cgagacttgt 180
gggcatatcg acctctatct cgaaagccgt gcccgcggct cgacgaagtg a 231
<210> 3
<211> 65
<212> PRT
<213> Artificial
<220>
<223>Rv0609B (- | 704475-704672 |) gene theory coded product amino acid sequence
<400> 3
Val Thr Asp Glu Lys Cys Val Arg Cys Gly Gly Asp Gln Leu Val Glu
1 5 10 15
Gly Ala Val Val Trp Asn Ala Pro Leu Arg Phe Lys Arg Glu Gly Ala
20 25 30
Gly His Phe Asn Arg Gly Thr Gln Val Asn Ala Val Ala Cys Glu Thr
35 40 45
Cys Gly His Ile Asp Leu Tyr Leu Glu Ser Arg Ala Arg Gly Ser Thr
50 55 60
Lys
65
<210> 4
<211> 18
<212> DNA
<213> Artificial
<220>
<223>F primer sequences
<400> 4
gaccgacgag aagtgcgt 18
<210> 5
<211> 18
<212> DNA
<213> Artificial
<220>
<223>R primer sequences
<400> 5
ggcacggctt tcgagata 18
<210> 6
<211> 18
<212> PRT
<213> Artificial
<220>
<223>Leakage annotation peptide fragment
<400> 6
Cys Gly Gly Asp Gln Leu Val Glu Gly Ala Val Val Trp Asn Ala Pro
1 5 10 15
Leu Arg
Claims (10)
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. the 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
Mycobacterium tuberculosis H37Rv encoding gene described in the claim 1 of cls gene.
A 4. species-specific PCR primers, for expanding the Mycobacterium tuberculosis H37Rv encoding gene described in claim 1.
5. the PCT primers described in claim 4, it is characterised in that the sequence of the primer is:
F:5’-GACCGACGAGAAGTGCGT-3’;
R:5’-GGCACGGCTTTCGAGATA-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 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 the 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’-GACCGACGAGAAGTGCGT-3’;
R:5’-GGCACGGCTTTCGAGATA-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 the Mycobacterium tuberculosis H37Rv encoding gene described in claim 1 and/or right
Seek the Specific PCR primers described in 4.
10. the Mycobacterium tuberculosis H37Rv encoding gene described in claim 1 is in tuberculosis epidemiology survey and/or clinical knot
Application in core patient quick diagnosis and discriminating.
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