CN113174443B - Mycobacterium identification method and biological material thereof - Google Patents
Mycobacterium identification method and biological material thereof Download PDFInfo
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Abstract
The invention relates to a method for identifying mycobacterium and a biological material thereof. Wherein, the chvd gene is applied to the identification of the strains of the mycobacteria, and the chvd gene can be obtained by amplifying a universal primer pair comprising a forward primer and a reverse primer. The universal primer pair includes a forward primer and a reverse primer, which are used to amplify the chvd gene of mycobacteria. The species identification method of the mycobacterium utilizes the universal primer pair to carry out PCR amplification to obtain chvd gene segments of the strains to be detected, and the chvd gene segments are compared after sequencing to identify the species of the strains to be detected. The classification and identification kit for mycobacteria uses chvd gene to identify mycobacteria species, and includes PCR technology for directly detecting chvd gene or other inspection technology based on the detection technology. The invention has accurate and reliable identification result, simple method and high identification speed for the species classification of the mycobacteria; the universal primer pair can identify the most common clinical isolates of nontuberculous mycobacteria.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to the field of molecular identification of microorganisms; in particular to a mycobacterium identification method, a kit, a universal primer pair and application of chvD gene.
Background
At present, tuberculosis remains a chronic infectious disease seriously endangering human health, and is the disease with the most number of deaths in a single infectious disease. The World Health Organization (WHO) has recent worldwide tuberculosis report data showing: about 1000 million new tuberculosis patients are discovered in 2019 worldwide, and the estimated tuberculosis death number is about 140 million. Nontuberculous Mycobacteria (NTM) refers to Mycobacteria other than Mycobacterium Tuberculosis Complex (MTC) and Mycobacterium leprae. More than 200 kinds of non-tuberculous mycobacteria are separated at present and are mostly pathogenic bacteria. In recent years, with the increasing and widespread of bacterial isolation technology, the occurrence of NTM infection and related diseases has been on the rise due to the change of infectious flora of immunocompromised hosts and the like, and it has been reported in our country that the proportion of NTM isolates in culture positive specimens increases from 4.3% in 1979 to 22.9% in 2010. According to the current trend of NTM epidemiology, with the increasingly appearing effect of national tuberculosis prevention and treatment planning, the ratio of NTM in the total tuberculosis morbidity is gradually increased. Therefore, the incidence of NTM in the future in China is expected to have room for improvement. Therefore, the research on NTM should also be highly regarded.
Clinical symptoms and signs of the NTM lung disease are very similar to those of the pulmonary tuberculosis, and characteristic symptoms are lacked, but systemic toxic symptoms such as low-heat emaciation and the like are lighter than the pulmonary tuberculosis. The main clinical manifestations include cough, expectoration, hemoptysis, chest pain and other respiratory symptoms, and also have general symptoms of hypodynamia, emaciation and the like. Therefore, in the absence of species identification, NTM disease is often misdiagnosed as tuberculosis. At present, no specific medicine is available for treating NTM lung diseases, treatment schemes of different strains are greatly different, and most NTM has poor curative effect on anti-tuberculosis medicine resistance. There are more than 30 kinds of common NTM capable of causing diseases, and there are great differences in drug selection aiming at different kinds of NTM treatment. Therefore, clinically, whether the patient has NTM infection or not and what kind of NTM infection is caused can be quickly and accurately determined, and the method has important significance for establishing an effective treatment scheme.
Currently, the methods for identifying NTM, which are commonly used in clinical practice, are divided into two main categories: one is the traditional biochemical method, which relies on a series of tedious biochemical experiments, taking weeks for slow growing mycobacteria. Moreover, the results of some biochemical experiments are sometimes ambiguous and difficult to accurately interpret. In addition, different NTM strains have similar phenotypes in biochemical experiments, which brings huge challenges for identifying the strains in the traditional biochemical experiments; the other is a molecular biology method for identifying NTM to species level by comparing differences of homologous genes/sequences, which is simple to operate, can quickly and accurately identify mycobacteria to species level, not only shortens identification time, but also improves discrimination between some mycobacteria. And various commercial kits (such as linear probe hybridization technology, DNA chip technology and multicolor fluorescence dissolution curve technology) have been derived for clinical application. In daily work, when sequence alignment is carried out by means of the existing strain identification marks such as 16S rRNA, hsp65, rpoB,16-23S rRNA and the like, strains with sequences which are different from known strains are often found, and accurate identification of the strains is difficult to carry out. Thus, more molecular markers are required and it is possible to classify the same NTM into more subspecies. This more detailed typing is not only helpful for epidemiological studies, but also beneficial for establishing the association of different subspecies with the onset of disease in patients, and thus understanding the host's immune and pathogenesis, similar studies have not been internationally focused on. Therefore, more mycobacterium species identification molecular markers are found, and the method has important reference value for improving identification capability and clinical diagnosis and treatment.
Disclosure of Invention
The technical problem to be solved by the invention is how to simply and rapidly identify the mycobacterium strains.
In order to solve the above technical problems,
the invention claims the use of a substance that specifically binds to the chvd gene of mycobacteria for the preparation of a product for identifying or aiding in the identification of mycobacteria.
The substance specifically combined with the chvd gene of the mycobacterium is a primer pair for specifically amplifying the chvd gene.
The primer pair consists of a forward primer and a reverse primer, wherein the forward primer is a single-stranded nucleotide shown in a sequence 1, and the reverse primer is a single-stranded nucleotide shown in a sequence 2.
The product is a reagent or a kit for identifying or assisting in identifying mycobacteria.
A primer pair for identifying or assisting in identifying mycobacteria is a primer pair for specifically amplifying chvd genes of the mycobacteria.
The primer pair consists of a forward primer and a reverse primer, wherein the forward primer is a single-stranded nucleotide shown in a sequence 1, and the reverse primer is a single-stranded nucleotide shown in a sequence 2.
The invention provides a kit for preparing and identifying or assisting in identifying mycobacteria, and the kit comprises the primer pair.
The invention provides a non-diagnostic method for identifying or assisting in identifying mycobacteria, comprising the steps of:
1) taking genome DNA of a strain to be detected from a lung disease patient as a template, and performing PCR amplification by using the primer pair to obtain a PCR amplification product of the strain to be detected;
2) and sequencing and comparing the PCR amplification products of the strains to be detected so as to identify whether the strains to be detected are mycobacteria and/or which mycobacteria exist.
And the comparison is to perform BLAST comparison on the sequence obtained by sequencing and the chvD gene sequences of all mycobacteria in a GenBank database, construct a phylogenetic tree and obtain an identification result.
The identification or assisted identification of the Mycobacterium as described hereinbefore allows the identification of which species of Mycobacterium the test strain belongs.
The test sample for identifying or aiding in the identification of a product of a mycobacterium is a sample originally from a patient with a lung disease.
The lung disease mentioned above represents a disease of the lung.
The application of the chvD gene in preparing products for identifying or assisting in identifying mycobacteria is also within the protection scope of the invention, and the chvD gene is the chvD gene of the mycobacteria.
The species classification identification result of the mycobacterium is accurate and reliable, the method is simple, and the identification speed is high; the universal primer pair identifies the vast majority of common clinical isolates of nontuberculous mycobacteria.
Drawings
FIG. 1 is a phylogenetic tree constructed by using the chvD gene for different standard strains of nontuberculous mycobacteria according to the present invention.
FIG. 2 shows the result of multiple sequence rearrangements of the test strain 34 and different species of Mycobacterium standard strain using the chvD gene.
FIG. 3 is a phylogenetic tree constructed by using the chvD gene of the present invention for the test strain 34 and different species of standard strains of mycobacteria.
FIG. 4 shows the result of multiple sequence rearrangements of the test strain 58 and different species of Mycobacterium standard strain using the chvD gene according to the present invention.
FIG. 5 shows a phylogenetic tree constructed by the chvD gene of the invention for the test strain 58 and different species of standard strains of mycobacteria.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The 58 standard strains of mycobacteria in the following examples were: mycobacterium marinum (M.marinum) ATCC 927, Mycobacterium fortuitum (M.fortuitum) ATCC6481, Mycobacterium phlei (M.pherei) ATCC11758, Mycobacterium kansasii (M.kansasii) ATCC12478, Mycobacterium intracellulare (M.intercellulare) ATCC 13950, Mycobacterium exotica (M.peregrinum) ATCC14467, Mycobacterium gordonii (M.gordonae) ATCC14470, Mycobacterium microflavus (M.flavescens) ATCC14474, Mycobacterium vaccae (M.vaccae) ATCC15483, Mycobacterium gastri (M.gastroli) ATCC15754, Mycobacterium terrestris (M.terrestris) ATCC 15755, Mycobacterium tuberculosis bovis (M.boviis) ATCC19210, Mycobacterium bufonis (M.paradoxi) ATCC 19250, Mycobacterium fortunei (M.diracharii) ATCC 19640, Mycobacterium phlei (M.19632), Mycobacterium xenopi (M.1953527, Mycobacterium phlei (M.faecalis) ATCC 3527, Mycobacterium phlei (M.19532, Mycobacterium phlei) ATCC 3527, Mycobacterium phlei (M.3, Mycobacterium phlei) ATCC 3523, Mycobacterium phlei (M.3, Mycobacterium phlei) Mycobacterium phlei (M.32, Mycobacterium phlei (M.phlei) Mycobacterium phlei (M.3, Mycobacterium phlei) ATCC 3523, Mycobacterium phlei (M.3, Mycobacterium phlei (M.32, Mycobacterium phlei) Mycobacterium phlei (M.32, Mycobacterium phlei (M.3, Mycobacterium phlei) Mycobacterium phlei (M.3, Mycobacterium phlei) Mycobacterium phlei (M.phlei) Mycobacterium phlei (M.32, Mycobacterium phlei) M.32, M.phlei (M.32, M.3, M.phlei (M.3, M.phlei (M.3, M.phlei (M.3, M.tuberculosis (M.3, M.tuberculosis (M.phlei (M.tuberculosis (M, mycobacterium minor (M.triviale) ATCC23292, Mycobacterium aureofaciens (M.aurum) ATCC23366, Mycobacterium tuberculosis H37Ra (M.tubericalis H37Ra) ATCC25177, Mycobacterium asia (M.asiaticum) ATCC25276, Mycobacterium avium subspecies avium (M.avium subsp.avium) ATCC25291, Mycobacterium africanum (M.africanum) ATCC25420, Mycobacterium neoformans (M.neoaurum) ATCC25795, Mycobacterium thuringiensis (M.szulgai) ATCC35799, Mycobacterium obuense (M.obuense) ATCC 27023, Mycobacterium lodorsalideae (M.rhodesiae) ATCC 27024, Mycobacterium chuanense (M.chauguense) ATCC27278, Mycobacterium aestivum (M.chrysogenum) ATCC 3568, Mycobacterium aestivum (M.ivoranense) ATCC 3568, Mycobacterium aestivum (M.tyarum) ATCC 3568, Mycobacterium avium (M.sourcense) ATCC 3568, Mycobacterium aureofaciens (M.3594, ATCC 3529. sakuwanensis) ATCC 3568, Mycobacterium avium (M.sakuchenoponse) ATCC 3513, ATCC 3533, Mycobacterium avium (M.sakuchenoponse) ATCC 3513, ATCC 3555, M.farinaceus) ATCC 3555, M.farinaceus, ATCC 3533, M.farinaceus, M.farinaceous (M.farinaceus) ATCC 3533, M.farinaceus, M.3533, ATCC 3533, M.farinaceus, ATCC 3533, M.farinaceus, ATCC 3533, M.farinaceus, ATCC 3533, M.farinaceous strain ATCC 3533, M.farinaceus, ATCC 3533, M.farinaceous strain (M.farinaceus, ATCC 3533, M.farinaceous strain (M.farinaceous strain ATCC 3533, M.farinaceous strain (M.3533, M.farinaceous strain (M.farinaceous strain) ATCC 3533, M.farinaceous strain (M.farinaceous strain ATCC 3533, M.farinaceous strain) ATCC 3533, M.farinaceous strain (M.farinaceous strain) ATCC 3533, M.farinaceous strain (M.farinaceous strain ATCC 3533, M.farinaceous strain ATCC 3533, ATCC 3578, M.farinaceous strain (M.farinaceous strain) ATCC 3533, M.farinaceous strain (M.farinaceous strain) ATCC 3578, M.farinaceous strain (M.farinaceous strain) ATCC 3533, M.farinaceous strain (M.farinaceous strain) ATCC 3578, ATCC 3533, M.farinaceous strain (M.farinaceous, mycobacterium chelonae (m.chelonae) ATCC14472, mycobacterium melitus (m.farcinenes) ATCC35753, mycobacterium senegalensis (m.senegalense) ATCC35796, mycobacterium flavum (m.gilvum) ATCC 43909, mycobacterium mucosus (m.mucogenium) ATCC49650, mycobacterium avium forest subsp.silvaticum) ATCC 49884, mycobacterium cryptum (m.ceratum) ATCC 51131, mycobacterium septicum (m.septicum) ATCC700731, mycobacterium curvatus (m.goodii) ATCC 700504, mycobacterium paratuberculorum (m.parascrofulum) ATCC BAA-614, mycobacterium cosmeticum (m.cosmetomicum) ATCC BAA-878, the above standard strains are all purchased at American type biological standard deposit (ATCC). In addition, chvD sequences of mycobacterium Marseillensis (M.Masssiliense) CCUG48898, mycobacterium abscessus borlei (M.bolletii) BD were from NCBI database.
The clinical isolates in the following examples were derived from the Beijing major disease clinical data and the sample resource library, tuberculosis library, of the Beijing thoracic Hospital affiliated with the university of capital medical science.
Example 1 amplification of the chvD Gene and establishment of a Trees of Standard strains of Mycobacterium
Chromosomal Virus D Gene (chvD) is one of mycobacteria (Mycobacterium) genome sequences, and according to the sequence, a universal primer pair capable of amplifying the Gene is designed and comprises a forward primer and a reverse primer, wherein the forward primer is 5'-TGCCCTCGAACCAGAACC-3' (SEQ ID No: 1), and the reverse primer is 5'-CTGCAGCGCTACGAGGAG-3' (SEQ ID No: 2). Wherein, the forward primer is positioned at the 96-113 position of the chvD gene (Rv2477) of the mycobacterium tuberculosis, and the gene bank number is NC-000962.3; the reverse primer is located at position 799-816 of the chvD gene of Mycobacterium tuberculosis, and the gene bank number is NC-000962.3.
The genomic DNA of 56 mycobacteria in the following table is used as a template and the universal primer pair is used as a primer for PCR amplification. The specific operation of PCR amplification is as follows: 25 mul of amplification reaction system, 20pmol of forward primer concentration and 20pmol of reverse primer concentration, 50ng of template DNA, 1U of Taq enzyme, 250 mu M of deoxynucleotide triphosphate final concentration and 1.5mM of MgCl2 final concentration; the amplification reaction conditions are as follows: denaturation at 95 ℃ for 5 min, followed by 35 cycles (95 ℃ for 30 sec, 56 ℃ for 30 sec, 72 ℃ for 30 sec), and extension at 72 ℃ for 5 min.
Sequencing the amplification product; and performing BLAST comparison on the sequence determination result and all chvD gene sequences of mycobacteria in a GenBank database to construct a phylogenetic tree, obtaining an identification result, wherein the matching degree is more than 97 percent to indicate that the matching is successful.
The mycobacterium identification method of the invention can accurately distinguish the strains which can not be clearly identified by phenotype typing and 16S rRNA gene typing, and comprises mycobacterium avium and mycobacterium intracellulare, mycobacterium kansasii and mycobacterium gastri, mycobacterium abscessus, mycobacterium cheloniae and the like. The mycobacterium abscessus complex includes mycobacterium abscessus and mycobacterium mosaic and m.bollitii, whose 16s rRNA encoding gene, 16s-23s rRNA Interregion (ITS), hsp65 and rpoB gene sequences are almost identical. And the similarity of the ChvD gene of the mycobacterium abscessus and the Mycobacterium mosaici is 97.5 percent, the similarity of the mycobacterium abscessus and the chvD of the M.bollitii is 97.8 percent, and the similarity of the mycobacterium mosaici and the chvD of the M.bollitii is 96.7 percent, so the chvD can be used for distinguishing the strains of the mycobacterium abscessus complex; the 16s rRNA coding gene of some mycobacteria (such as M.celatum and M.terrae) is multi-copy, but the ChvD gene is single-copy, so that the difficulty in judging the strain identification result due to multi-copy of the target gene can be avoided.
The sequence similarity of a to-be-detected strain of the mycobacterium intracellulare and a standard strain is 98.3% -100%, the sequence similarity of a to-be-detected strain of the mycobacterium avium and the standard strain is 99.1% -100%, the sequence similarity of the to-be-detected strain of the mycobacterium abscessus and the standard strain is 97.9% -100%, the sequence similarity of the to-be-detected strain of the mycobacterium kansasii and the standard strain is 98.8% -100%, the sequence similarity of the to-be-detected strain of the mycobacterium gordonii and the standard strain is 97.9% -100%, the sequence similarity of a to-be-detected strain of the adventitious mycobacterium and the standard strain is 97.8% -100%, and the sequence similarity of the to-be-detected strain of the mycobacterium tuberculosis and the standard strain is 99.8% -100%. Therefore, the chvD gene can be used for identifying the strains of the mycobacteria, and can identify most common non-tuberculous mycobacteria by taking the similarity of the sequence of the chvD gene and the sequence of a standard strain as a boundary, which is greater than 97%.
Example 2 identification of clinical isolate 34
Extracting genome DNA of the clinical isolate, carrying out PCR amplification by using a universal primer pair as a primer according to the method in the embodiment 1, and sequencing the amplification result, wherein the sequence of the amplification result is shown as SEQ ID No. 3 in a sequence table. The gene chvD after amplification of the clinical isolate to be identified was subjected to multiple sequence rearrangement with the gene chvD of the above 58 standard strains by CLUSTAL2.1 software, and the similarity to the standard strains (similarity to M.intracellulare standard strain 100%) was compared, and was preliminarily identified as M.intracellulare (as shown in FIG. 2).
Further alignment with the evolutionary trees of various standard strains of mycobacteria established in example 1 confirmed that they were M.intracellulare (as shown in FIG. 3).
EXAMPLE 3 identification of clinical isolate 58
Extracting genome DNA of the clinical isolate, carrying out PCR amplification by using a universal primer pair as a primer according to the method in the embodiment 1, and sequencing the amplification result, wherein the sequence of the amplification result is shown as SEQ ID No. 4 in a sequence table. The gene chvD after amplification of clinical isolates to be identified is subjected to multiple sequence rearrangement with the gene chvD of the 58 standard strains by using CLUSTAL2.1 software, and the similarity with the standard strains (the similarity with the standard strains of the mycobacterium africanum is 100 percent) is compared, so that the gene chvD is preliminarily identified as the mycobacterium africanum and belongs to the mycobacterium tuberculosis complex (as shown in figure 4).
Further comparison with the evolutionary trees of various standard strains of Mycobacterium established in example 1 confirmed that they are M.africanum (as shown in FIG. 5).
Meanwhile, the strains of example 2 and example 3 were identified as M.intracellulare and M.tuberculosis by sequencing of the 16s rRNA-encoding gene, 16s-23s rRNA spacer region (ITS), hsp65, and rpoB gene, respectively.
The method for identifying mycobacteria and the biological material thereof of the present invention are more advantageous for commonly used species that cannot be distinguished by 16srRNA coding gene, 16s-23srRNA Interregional (ITS), hsp65 and rpoB gene, etc., as described above: the 16srRNA coding gene, the 16s-23s rRNA Interregional (ITS), the hsp65 and the rpoB gene sequences of the mycobacterium abscessus complex are almost completely the same, and the similarity of the ChvD gene is 96.7-97.8 percent, so that the mycobacterium abscessus complex can be used for distinguishing three subspecies of the abscess complex; some mycobacteria (e.g. m.celatum and m.terrae) have multiple copies of the 16s rRNA coding gene but a single copy of the ChvD gene, which avoids difficulties in determining species identification due to multiple copies of the target gene.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Sequence listing
<110> Beijing thoracic Hospital affiliated to capital medical university
Wuhan city pneumology hospital (Wuhan city tuberculosis prevention and cure station)
<120> method for identifying mycobacterium and biological material thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 666
<212> DNA
<213> M.intracellulare
<400> 3
ctccacgcca ggatgtgggt gcaggtgcgg tccaggaacc accggtcgtg cgaaatcacc 60
accgcgcagc cggggaactg ctcgagggcg ttctccagcg agctcagggt ttcgacgtcg 120
aggtcgttgg tgggctcgtc gagcaggatg aggtttccgc cctgcttgag cgtcagcgcg 180
aggttgagcc ggttgcgctc gccgcccgag agaacgccgg ccggcttctg ctggtctggg 240
cccttgaacc cgaacgccga cacgtaggcc cgcgacggca cctcggtctg gccgacgacg 300
atgtggtcca gcccgtcgga gacgacctcc cagacgttct tcttcggatc gatgcccgcg 360
cgggtctggt cgacgtagct cagcttgacc gtctcgccga ccttgaccgt gccgctgtcc 420
ggctcctcga gaccgacgat ggttttgaac agcgtggtct ttccgacgcc gttggggccg 480
atgaccccga cgatgccgtt gcgcggcagg gtgaacgaca ggtccttgat gagggtgcgc 540
ccgccgtagc ccttgtcgag gtgctcgacc tcgaccacca cgttgcccag ccgcggaccg 600
accgggatct ggatctcctc gaagtcgagc ttgcgcgtct tctcggcctc ggcggccatc 660
tcccga 666
<210> 4
<211> 666
<212> DNA
<213> M.africanum
<400> 4
ctccacgcca ggatgtgcgt gcacgtgcgg tcaagaaacc agcgatcgtg cgaaatcacc 60
acggcgcagc cggggaagtt cagcagagca ttctccagcg aacccagagt ctcgacatcc 120
aggtcgttcg tcggttcgtc gagcagaatc aggttgccgc cctgtttgag cgtcaacgca 180
aggttgagcc tgttgcgctc cccgccggat agcacaccgg ccggtttttg ctggtccggt 240
cccttaaacc cgaatgccga cacgtaggcc cgtgacggca cttcggtttg accgacctgg 300
atatagtcca gaccgtccga gacaacctcc cagacggtct tccgcggatc gatgccagca 360
cgggcctggt ccacgtaact cagcttgacg gtctcgccga ccttgacgct gccgctgtcc 420
ggtgtctcga gcccgacgat ggttttgaac agtgtggtct tgcctacccc gttgggccca 480
atgacgccga cgatgccatt gcggggcaag ctgaacgaca ggtccttgat cagggcgcgc 540
ccgtcgtagc ccttatcgag gtggtcgacc tcaaccacca cgttgcctag gcggggcccg 600
accgggatct gaatctcctc gaagtcgagc ttgcgggtct tctccgcctc ggctgccatc 660
tcccga 666
Claims (5)
1. The application of the primer pair for specifically amplifying the chvd gene of the mycobacterium in preparing products for identifying or assisting in identifying the mycobacterium is characterized in that the primer pair consists of a forward primer and a reverse primer, wherein the forward primer is a single-stranded nucleotide shown in a sequence 1, and the reverse primer is a single-stranded nucleotide shown in a sequence 2.
2. The use according to claim 1, wherein the product is a reagent or kit for identifying or aiding in the identification of mycobacteria.
3. A primer pair for identifying or assisting in identifying mycobacteria, which is characterized in that the primer pair is a primer pair for specifically amplifying chvd gene of mycobacteria; the primer pair consists of a forward primer and a reverse primer, wherein the forward primer is a single-stranded nucleotide shown in a sequence 1, and the reverse primer is a single-stranded nucleotide shown in a sequence 2.
4. A kit for identifying or aiding in the identification of mycobacteria, comprising the primer pair of claim 3.
5. A non-disease diagnostic method for identifying or aiding in the identification of mycobacteria comprising the steps of:
1) performing PCR amplification by using the primer pair of claim 3 using genomic DNA of a test strain from a lung disease patient as a template to obtain a PCR amplification product of the test strain;
2) sequencing and comparing PCR amplification products of the strains to be detected so as to identify whether the strains to be detected are mycobacteria and/or which mycobacteria;
and the comparison is to carry out BLAST comparison on the sequence obtained by sequencing and all chvD gene sequences of mycobacteria in a GenBank database, construct a phylogenetic tree and obtain an identification result.
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| CN102634575B (en) * | 2012-03-26 | 2014-04-02 | 中国人民解放军第三O九医院 | Rapid identification method and kit of novel mycobacterium strain |
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| CN106834498A (en) * | 2017-03-03 | 2017-06-13 | 广州金域医学检验中心有限公司 | The primer and method of a kind of Rapid identification mycobacteria |
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