CN113025729A - Mycobacterium tuberculosis drug-resistance-related gene mutation site and application thereof - Google Patents
Mycobacterium tuberculosis drug-resistance-related gene mutation site and application thereof Download PDFInfo
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Abstract
The invention provides a mycobacterium tuberculosis drug resistance related gene mutation site and application thereof, wherein the gene mutation site is metH gene mutation site and/or rv3253c gene promoter region mutation site; the metH gene mutation site and its mutation type are A1108G, T1631C or A3292C with respect to the nucleic acid having the sequence of SEQ ID No. 1; relative to the nucleic acid with the sequence of SEQ ID No.2, the mutation site and the mutation type of the promoter region of the rv3253C gene are G7A, C31T, C33T, G35A, T39C, G40A, G42A or G57A. The mutation on the metH gene or the rv3253c gene promoter region provided by the invention can increase the drug resistance level of the mycobacterium tuberculosis PAS, so that the gene mutation site related to the drug resistance of the mycobacterium tuberculosis PAS provided by the invention can be used as a detection target to be applied to the detection of the drug resistance of the mycobacterium tuberculosis PAS, the drug resistance positive detection rate is improved, the diagnosis time of corresponding patients can be effectively shortened, and the treatment time and cost of the patients are saved.
Description
Technical Field
The invention relates to the technical field of mycobacterium tuberculosis drug-resistant gene mutation detection, in particular to a mycobacterium tuberculosis drug-resistant related gene mutation site for aminosalicylic acid (PAS) and application thereof.
Background
Tuberculosis is an infectious disease caused by infection of mycobacterium tuberculosis, and is the first fatal infectious disease worldwide. The increasing multidrug-resistant tuberculosis is one of the most serious challenges facing the prevention and control of tuberculosis worldwide. Compared with non-drug resistant tuberculosis, the treatment time of the drug resistant tuberculosis is increased from 6 months to 12-24 months, and the treatment cost is increased by tens of times. China is a drug-resistant tuberculosis big country, and the number of patients with drug-resistant tuberculosis in China accounts for 13 percent of the whole world. In recent years, due to the increase of multidrug-resistant tuberculosis and pan-drug-resistant tuberculosis, aminosalicylic acid (PAS) is mainly used for strengthening treatment schemes of patients with pan-drug-resistant tuberculosis and patients with multidrug-resistant tuberculosis who do not respond to treatment. The side effect of PAS is mainly the gastrointestinal side effect which is difficult to tolerate, but the powerful antituberculosis effect is indispensable for curing drug-resistant tuberculosis, so that the understanding of the drug resistance of patients before the drug is taken is particularly important.
The phenotype drug sensitivity test has high requirements on personnel, infrastructure and biological safety, and is time-consuming and labor-consuming, and the conventional culture method at least needs additional 3-4 weeks to obtain drug sensitivity results. Molecular resistance diagnosis has been recommended by WHO guidelines because of its rapid characteristics. For tuberculosis patients, especially drug-resistant tuberculosis patients, rapid diagnosis of drug resistance is important for selection and formulation of clinical protocols.
The currently known mechanism of action of PAS is interference with folic acid metabolism and thymidylate nucleotide biosynthesis of Mycobacterium tuberculosis. The resistance mechanism mainly involves 6 genes (thyA, dfrA, folC, folP1, ribD, thyX) in the above pathway. However, the mutation of the above 6 genes can only explain less than 40% of the phenotypic drug resistance, and diagnosis of drug resistance using only the above genes has a high false negative rate. Considering that the conventional detection does not carry out the detection of PAS drug sensitivity and related drug resistance genes, the discovery of the new drug resistance genes and the detection method of PAS has important significance for the rapid diagnosis and treatment of drug-resistant tuberculosis.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a mycobacterium tuberculosis PAS drug resistance related gene mutation site and application thereof, wherein the gene mutation site specifically comprises the following components: compared with the metH gene of the wild type H37Rv standard strain, the 1108 th (base a is mutated into g) or 1631 th (base t is mutated into c) or 3292 th (base a is mutated into c) mutation site of the metH gene; compared with a wild type H37Rv standard strain, the-7 th (the base g is mutated into a) mutation site or the-31 th (the base c is mutated into t) mutation site or the-33 th (the base c is mutated into t) mutation site or the-35 th (the base g is mutated into a) mutation site or the-39 th (the base t is mutated into c) mutation site or the-40 th (the base g is mutated into a) mutation site or the-42 th (the base g is mutated into a) mutation site or the-57 th (the base g is mutated into a) mutation site of the rv3253c gene promoter region.
The invention adopts the following technical scheme for solving the technical problems:
the first aspect of the invention is to provide a mycobacterium tuberculosis PAS drug resistance related gene mutation site, the gene mutation site is metH gene and/or rv3253c gene promoter region mutation site; relative to the nucleic acid with the sequence of SEQ ID No.1, the MetH gene mutation site and the mutation type thereof are A1108G, T1631C or A3292C; relative to the nucleic acid with the sequence of SEQ ID No.2, the mutation site of the promoter region of the rv3253C gene and the mutation types thereof are G7A, C31T, C33T, G35A, T39C, G40A, G42A or G57A.
The second aspect of the present invention provides the use of the gene mutation site provided in the first aspect in the preparation of a PAS-resistant detection reagent for mycobacterium tuberculosis, the detection reagent comprising a primer composition and/or a probe for detecting the gene mutation site.
Further, the primer composition comprises a primer with a nucleotide sequence of SEQ ID No. 3-6 and/or a primer with a nucleotide sequence of SEQ ID No. 7-8.
Further, the sequence of the probe is selected from SEQ ID No.9 to SEQ ID No.14 and the base complementary sequence thereof.
The third aspect of the present invention provides an application of the gene mutation site provided in the first aspect in the preparation of a PAS-resistant mycobacterium tuberculosis detection kit, which comprises a primer composition and/or a probe for detecting the gene mutation site.
Further, the primer composition comprises a primer with a nucleotide sequence of SEQ ID No. 3-6 and/or a primer with a nucleotide sequence of SEQ ID No. 7-8; the sequence of the probe is selected from SEQ ID No. 9-SEQ ID No.14 and the base complementary sequence thereof
The fourth aspect of the present invention provides a PAS drug-resistant Mycobacterium tuberculosis detection kit, which comprises a primer composition and/or a probe for detecting the gene mutation site provided in the first aspect.
Further, the primer composition comprises a primer with a nucleotide sequence of SEQ ID No. 3-6 and/or a primer with a nucleotide sequence of SEQ ID No. 7-8; the sequence of the probe is selected from SEQ ID No. 9-SEQ ID number 14 and the base complementary sequence thereof.
The fifth aspect of the present invention is to provide a rapid screening method for drug-resistant mutant base of mycobacterium tuberculosis using the detection kit of the fourth aspect, the rapid screening method being amplifying metH gene and its internal base sequence and detecting mutant site of metH gene; and/or amplifying the rv3253c gene promoter region and detecting the mutation site of the rv3253c gene promoter region.
The sixth aspect of the invention provides a second generation sequencing method for detecting drug-resistant mutant bases of mycobacterium tuberculosis, which comprises the steps of carrying out high-throughput sequencing, comparing with a metH gene with a nucleotide sequence of SEQ ID No.1 and/or an rv3253c gene promoter region with a nucleotide sequence of SEQ ID No.2, and detecting whether base mutation occurs at 1108 th, 1631 th or 3292 th positions of the metH gene and the base sequence thereof and/or whether base mutation occurs at 7 th, 31 th, 33 th, 35 th, 39 th, 40 th, 42 th or 57 th positions of the rv3253c gene promoter region and the base sequence thereof.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the mutation on the metH gene or the rv3253c gene promoter region provided by the invention can cause the increase of the drug resistance level of the mycobacterium tuberculosis PAS, so that the gene mutation site related to the drug resistance of the mycobacterium tuberculosis PAS provided by the invention can be used as a detection target to be applied to the detection of the drug resistance of the mycobacterium tuberculosis PAS, the drug resistance positive detection rate is improved, the diagnosis time of the part of patients can be effectively shortened, and the treatment time and cost of the patients are saved.
Drawings
FIG. 1 is a diagram showing the positions of mutation sites in metH gene (FIG. A) and promoter region (FIG. B) of rv3253c gene of Mycobacterium tuberculosis in one embodiment of the present invention;
FIG. 2 is a nucleotide and amino acid sequence of a metH gene mutation in one embodiment of the present invention.
Detailed Description
The present inventors observed a change of PAS-resistant phenotype in a propagation cluster with very small genetic difference by a whole genome sequencing method, and found a mutation of methionine synthase (rv2124c, metH) by comparing only several different SNPs between PAS-resistant strains and sensitive strains within the cluster, and subsequently, the inventors expanded the sample size and examined the samples in 28 PAS-resistant strains and 107 PAS-sensitive strains, and found mutations of the metH gene in 4 PAS-resistant strains, respectively 1108a > g, 1631t > c, and 3292a > c, with a frequency of occurrence of the above mutations being 14.3%, and did not find a mutation of the metH gene in the sensitive strains, which indicates that there is a certain frequency of occurrence of a mutation of the metH gene in PAS-resistant strains. Meanwhile, the inventor obtains a PAS drug-resistant strain in an in-vitro drug-resistant induction mode, carries out whole genome sequencing on the drug-resistant induction strain and a sensitive parent strain, and finds that the rv3253c gene promoter region in the drug-resistant induction strain has unique differential mutation and more mutation types by comparing the differential SNP through data analysis, which shows that the rv3253c gene promoter region mutation is related to PAS drug resistance. Therefore, the mutation of metH gene or rv3253c gene promoter can be used as the diagnostic basis for clinical PAS drug-resistant molecular diagnosis.
Based on the gene, the invention provides a mycobacterium tuberculosis PAS drug resistance related gene mutation site and application thereof, wherein the gene mutation site specifically comprises the following components: compared with the metH gene (SEQ ID No.1) of the wild type H37Rv standard strain, the 1108 th (base a is mutated into g) or 1631 th (base t is mutated into c) or 3292 th (base a is mutated into c) mutation site of the metH gene; compared with the rv3253c gene promoter region (SEQ ID No.2) of a wild type H37Rv standard strain, the-7 th (base g is mutated into a) mutation site or the-31 th (base c is mutated into t) mutation site or the-33 th (base c is mutated into t) mutation site or the-35 th (base g is mutated into a) mutation site or the-39 th (base t is mutated into c) mutation site or the-40 th (base g is mutated into a) mutation site or the-42 th (base g is mutated into a) mutation site or the-57 th (base g is mutated into a) mutation site of the rv3253c gene promoter region of the rv3253c gene promoter region.
The present invention will be described in detail and specifically with reference to the following examples and drawings so as to better understand the present invention, but the following examples do not limit the scope of the present invention.
In the examples, the conventional methods were used unless otherwise specified, and the reagents used were, for example, conventional commercially available reagents or reagents prepared by conventional methods without specifically specified.
Example 1
In this example, the specific operation method and results for screening the metH gene mutation site related to PAS drug resistance are as follows:
step one, predicting the base mutation site related to PAS drug resistance
Firstly, adopting a second generation sequencing technology to carry out whole genome sequencing on 55 multi-drug-resistant mycobacterium tuberculosis clinical strains from China, and predicting base mutation sites related to drug resistance of aminosalicylic acid (PAS) by a bioinformatics method. Single Nucleotide Polymorphism (SNP) analysis was performed using H37Rv (NC000962.3) as an alignment template, and a clade was constructed based on the SNPs. The change of PAS-resistant phenotype was observed in a spread cluster with very small genetic difference, and the difference SNP between the PAS-resistant strain and the PAS-sensitive strain in the cluster was compared, and the metH gene (rv2124c) mutation was found.
Step two, further verifying the predicted base sites related to PAS drug resistance
The sample size was expanded and the prediction results of metH gene and its mutation were further verified. 135 clinical strains were selected and subjected to a phenotypic drug susceptibility test of PAS, and among them, 28 strains were PAS-resistant strains and 107 strains were PAS-sensitive strains.
As shown in fig. 1A, 2 and table 1, 4 strains generated base mutation in the metH gene, including 1108 th (base a mutation is g), 1631 th (base t mutation is c), 2 th and 3292 th (base a mutation is c), the mutation rate was 14.3% (the mutation rates at the above positions are 3.6%, 7.1% and 3.6%, respectively), and none of the metH genes of 107 sensitive strains were mutated. It is further proved that the predicted gene metH and the base mutation site thereof are possibly related to the drug resistance of the Mycobacterium tuberculosis PAS.
TABLE 1 mutation site of metH Gene in PAS drug-resistant Strain
| Position in genome | Nucleotide positions and changes | Amino acid position and changes |
| 2384988 | a1108g | C370R |
| 2384445 | t1631c | Q544R |
| 2382784 | a3292c | Y1098D |
Step three, comparing and analyzing the mutation rate of the metH gene and the PAS known drug resistance related gene
PAS is known that drug resistance related genes are folic acid metabolism, thyA, dfrA, folC, folP1, ribD and thyX in a DNA synthesis pathway, and are commonly applied to detection of drug resistance of PAS of mycobacterium tuberculosis. The mutation rate of the 6 genes is about 40 percent of that of a phenotypic drug-resistant strain, and the diagnosis of drug resistance by only using the genes has higher false negative rate.
Sequencing analysis is carried out on the 6 genes in the 28 PAS drug-resistant mycobacterium tuberculosis strains in the step two, and the mutation rate of more than 11 strains is found to be 39.3 percent.
If the detection of metH gene mutation is added, the mutation rate of PAS drug resistance detection can reach 53.6 percent, and the accuracy can be improved by 14.3 percent.
The analysis result shows that the mutant bases of the Single Nucleotide Polymorphism (SNP) site, position 1108 (the base a is mutated into g), position 1631 (the base t is mutated into c) and position 3292 (the base a is mutated into c) of metH can be used as a marker for PAS drug resistance molecular diagnosis of the mycobacterium tuberculosis, and the detection rate of PAS drug resistance mycobacterium tuberculosis is favorably improved.
Example 2
In this example, the gene mutation site of the rv3253c promoter region related to PAS resistance was screened by a method of inducing resistance in vitro, and the specific procedures and results are as follows:
a Mycobacterium tuberculosis model strain H37Ra with an appropriate concentration was spread on a 7H11 plate with PAS concentration of 1mg/mL, and after standing culture at 37 ℃ for 4-6 weeks, a single clone on the plate was picked up. After the expanded culture, the parent H37Ra wild strain and the obtained induced drug-resistant strain are subjected to whole genome sequencing, and the difference SNP is analyzed. The promoter mutation of rv3253c of 8 different types is found in 8 drug-resistant clones (as shown in fig. 1B), and the sites of the rv3253c promoter mutation of the 8 different types are respectively the-7 th (base g mutated to a) mutation site, the-31 th (base c mutated to t) mutation site, the-33 th (base c mutated to t) mutation site, the-35 th (base g mutated to a) mutation site, the-39 th (base t mutated to c) mutation site, the-40 th (base g mutated to a) mutation site, the-42 th (base g mutated to a) mutation site, and the-57 th (base g mutated to a) mutation site.
Example 3
This example demonstrates the effect of rv3253c promoter mutation on gene expression and overexpression of rv3253c gene on PAS MICs.
Firstly, verifying the influence of rv3253c promoter mutation on gene expression: the influence of promoter mutation on subsequent gene transcription is verified through a lacZ enzyme activity experiment. The lacZ expression plasmid carrying the promoter mutation is constructed, the plasmid is transferred into Mycobacterium Smegmatis (Mycobacterium Smegmatis), and the influence of the promoter mutation on transcription is verified by detecting the change of the lacZ enzyme activity. The specific operation method is as follows:
(1) sample preparation:
1. centrifuging 1ml of Mycobacterium smegmatis liquid (shaking 3 days at 37 ℃) at 13000rpm for 1min to remove the culture medium, and washing the cells with 500 μ l of 1 × PBS for 2 times;
centrifuging at 2.6000rpm for 3.5min, and removing the supernatant;
3. resuspend pellet with 50. mu.l 1 × lysis solution and store sample at 4 ℃;
4. samples were frozen on dry ice (30s) and then thawed in a 37 ℃ water bath (90s) in duplicate;
6. the supernatant was centrifuged at 4 ℃ for 5min at maximum speed (13000rpm) and transferred to a new EP tube.
(2) And (3) enzyme activity determination:
1. adding 1, 5 and 10 mul of cell lysate into a 96-well plate respectively;
2.96 well plates were loaded with 17. mu.l of ONPG and 50. mu.l of 1 clear buffer (mercaptoethanol added);
3. incubation in microplate reader at 37 ℃ for 30min and OD read every 5min420；
4. Stopping the reaction, adding 125 ul of stop solution, wherein the color may be deepened, and the final volume is 192 ul;
5. reading absorbance at 420nm, untransfected cell lysates as controls;
6. 3 different volumes of lysate (1, 5, 10. mu.l) were measured and the absorbance change should be linear;
7. after obtaining an accurate reading of the lysate, the protein lysate protein concentration is determined (BCA method) and the lysate enzyme activity is calculated as follows:
enzyme activity (nmol) amount of hydrolyzed ONPG/t/protein (mg);
the amount of ONPG hydrolyzed (nmol) ═ OD420)(1.92×105nL)/(4500 nL/nmol-cm)(1cm)；
(3) Determination of protein concentration by BAC method:
solution A: solution B is 50: 1 preparing a mixed solution;
2.180 mul of mixed solution and 20 mul of standard substance;
3.180 μ l mixture +10 μ l lysate +10 μ l ddH2O；
Incubating at 4.37 deg.C for 30min, and detecting on computer.
The experimental result shows that the mutation can improve the expression level of the rv3253c gene by 10-20 times.
Secondly, verifying the influence of over-expression of rv3253c gene on PAS MIC
An rv3253c gene overexpression plasmid based on polyG is constructed, introduced into a non-toxic model strain H37Ra, and verified to be successfully overexpressed by using real time-PCR. PAS MIC identification is carried out on the over-expression strain, and the MIC of the over-expression strain is found to be improved by more than 4 times.
The experiments prove that the mutation of the rv3253c promoter can increase the expression of a subsequent gene, namely the rv3253c gene, so that the drug resistance of the strain to PAS is improved.
Example 4
The embodiment provides a kit for detecting PAS drug resistance related gene mutation sites by adopting a PCR amplification-sequencing method, which can detect 1108 th (base a is mutated into g), 1631 th (base t is mutated into c) and 3292 th (base a is mutated into c) mutations of a metH gene, as well as-7 th (base g is mutated into a), -31 th (base c is mutated into t), -33 th (base c is mutated into t), -35 th (base g is mutated into a), -39 th (base t is mutated into c), -40 th (base g is mutated into a), -42 th (base g is mutated into a) and-57 th (base g is mutated into a) mutations of a rv3253c gene promoter region. The kit comprises one or a combination of several reagents:
(1) a reagent for extracting DNA from a sample to be tested;
(2) PCR primers and related PCR reaction reagents for amplifying metH gene of tubercle bacillus sample DNA, rv3253c gene promoter region;
(3) reagents for direct sequencing of PCR products.
Among them, PCR primers for detecting mutations in metH gene and rv3253c gene promoter region are shown in Table 2.
A method for detecting gene mutation of metH gene and rv3253c gene promoter region using the kit, comprising the steps of:
(1) collecting a cultured tubercle bacillus sample of an individual to be detected, and extracting whole genome DNA;
(2) using the extracted DNA as a template, and using primers (Table 2) designed by the invention aiming at the metH gene mutation and the rv3253c gene promoter region mutation to amplify the DNA sequence of the mutation segment to obtain a corresponding PCR amplification product;
(3) the obtained PCR product was subjected to primary sequencing, and the determined sequence was compared with the normal sequence of metH gene, rv3253c gene promoter region to determine whether a mutation existed: FIG. 2 shows, in boxes, the bases and amino acids mutated from the wild type a to g at base 1108, which mutations mutate the cysteine (C) at position 370 of the wild type MetH protein to arginine (R); the 1631 th base is changed from wild-type t to c, and the mutation makes the 544 th glutamine (Q) of the wild-type MetH protein mutated into arginine (R); the 3292 th base is guanine (g) from wild-type adenine (a), the mutation mutates tyrosine (Y) at position 1098 of the wild-type MetH protein into aspartic acid (D), and the mutation causes the function of the MetH protein to be changed, thereby causing the drug resistance of PAS. FIG. 2 is a box mark showing the base of mutation in the Rv3253c promoter region, the mutation results in increased expression of Rv3253c and thus PAS resistance, and the PAS phenotype of the strain containing the mutation is drug resistance.
(4) According to the above experimental results, whether the pathogenic bacteria of the patient is PAS resistant Mycobacterium tuberculosis strain caused by metH gene mutation or rv3253c promoter region mutation or not is analyzed.
The PCR product obtained by the above method can also be detected by other methods such as a DNA hybridization probe method. The probes can be hybridized with the mutant metH gene and the mutant rv3253c promoter region nucleotide sequence, or two probes can be hybridized with the normal or mutant metH gene and the rv3253c promoter region nucleotide sequence. The probe can be selected from an isotope label, a colored substance label or a fluorescent substance label. Among them, the probes designed by the present inventors are shown in Table 3. In addition, mutations can be determined by site-specific PCR primers, restriction endonucleases or single-strand conformation polymorphisms.
TABLE 2 sequence information of amplification primers for metH Gene, rv3253c promoter region
TABLE 3 Artificial Probe sequence information Table
The kit for detecting PAS drug-resistant mycobacterium tuberculosis provided by the invention is internally provided with a reagent for detecting gene mutation of a promoter region of a metH and/or rv3253c gene, and also provides manufacturing, using and selling information of related medicines or biological products which is audited by administrative drug administration. For example, the kit for detecting the gene mutation of the promoter region of the metH and/or rv3253c gene by using a PCR-direct sequencing method comprises one or more of amplification primers, dNTPs, DNA polymerase for PCR reaction, a buffer solution thereof and reagents required for sequencing. As known to those skilled in the art, the above components are only schematic components, and as the amplification primers are a pair of primers metH-F and metH-R provided in the present invention, the DNA polymerase used for the PCR reaction is an enzyme capable of performing PCR amplification.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications or alterations to this practice will occur to those skilled in the art and are intended to be within the scope of this invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
<110> university of Compound Dan
<120> mycobacterium tuberculosis drug resistance-related gene mutation site and application thereof
<160> 14
<170> SIPOSequenceListing 1.0
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gtgactgcgg ccgacaagca cctctacgac accgatctgc tcgacgtctt gtcgcagcga 60
gtgatggtcg gcgacggtgc aatgggaacc caactacagg ccgcggacct cacgctcgac 120
gacttccgcg gcctggaggg ctgcaacgag atcctcaacg aaacccgccc tgacgtgctg 180
gaaaccattc accgcaacta tttcgaagcg ggcgccgacg ccgtcgagac gaacacgttt 240
ggctgcaacc tgtccaacct cggcgactac gacatcgccg acaggatccg cgatctatca 300
cagaagggca ccgcgatcgc acgccgggtg gccgacgagc tgggcagtcc cgaccgcaag 360
cgctacgtgc tggggtcgat ggggccgggc accaagctgc cgactctggg ccacaccgaa 420
tacgcggtga tccgcgacgc ctacaccgag gccgcgctgg gcatgctgga cggcggagcc 480
gacgccatcc tggtggaaac ctgccaggac ctactgcagc tgaaggcggc ggtgttgggg 540
tcgcggcggg cgatgacgcg ggccgggcgg cacattccgg tgtttgccca cgtcaccgtc 600
gagaccaccg gcaccatgct gctgggcagc gagatcgggg cggcgttgac cgctgtcgag 660
ccgctcggtg tggacatgat cggcttgaac tgcgcgacgg gtccggccga gatgagcgag 720
cacctgcgcc acctgtcccg gcacgcccgc atcccggtgt cggtgatgcc caacgccggg 780
ttgccggtgc tgggcgccaa gggcgccgaa tatccgttgc tgcccgacga attggccgag 840
gcgctggccg gcttcatcgc cgagttcggg ctctcgctgg tcggtggctg ctgcggcacc 900
accccggccc atatccgcga agtggctgcc gcggttgcga acatcaagcg tcccgagcga 960
caggtcagct acgagccgtc ggtgtcgtcg ctgtacaccg caatcccgtt cgcccaggac 1020
gcctcggttc tggtgatcgg ggagcgaacg aacgccaacg gctccaaggg ttttcgtgag 1080
gcgatgatcg ccgaggacta ccagaagtgc ctggacatcg ccaaggacca gacccgcgac 1140
ggcgcccacc tgctggacct gtgtgtggac tacgtgggcc gcgacggtgt ggccgacatg 1200
aaggcgctgg ccagccggct ggccacgtcc tcgacgctgc cgatcatgct ggactccacc 1260
gaaaccgcgg tgctgcaggc gggtttggag catctgggtg gccgttgcgc gatcaactcg 1320
gtgaactacg aggacggcga cggcccggaa tcgcgctttg ccaagaccat ggcgctggtc 1380
gccgagcacg gcgcggcggt ggtcgcgctg accatcgacg aagagggcca ggcccgcacc 1440
gcgcagaaga aggtcgagat cgccgagcgg ctgatcaacg acatcaccgg caactggggc 1500
gtcgacgaat catccatcct catcgacacc ttgacgttca ccatcgccac cggtcaggag 1560
gagtcccgcc gcgacggcat cgagaccatc gaggcgatcc gcgaactgaa aaagcgccac 1620
ccggatgtgc agaccacact tggtctgtcc aacatctcgt ttggtctcaa tcccgcagcg 1680
cgccaggtgc tcaactcggt gttcctgcac gaatgccaag aagcggggct ggattcggcg 1740
atcgtgcacg cgtcgaagat cctgccgatg aaccggattc ccgaggagca acgcaacgtc 1800
gccctggatc tggtctacga ccgccgccgc gaggactacg atccgctgca ggagctgatg 1860
cggctgttcg aaggcgtgtc ggcggcctcc tcgaaagagg accgactggc tgaactagct 1920
gggctgccgc tgttcgaacg gctggcccaa cgcatcgtcg acggcgagcg caacggcctg 1980
gacgccgatc tcgacgaggc gatgacgcaa aagccgccgc ttcagatcat caacgaacat 2040
ctgctggccg gcatgaagac ggtcggcgag ctcttcggct ccggccagat gcagctgccg 2100
ttcgtgctgc agtcggcgga ggtaatgaaa gccgccgtcg cgtatctgga accgcacatg 2160
gagcgctcgg acgacgattc gggcaaggga cgcatcgtgc tggccaccgt caagggcgac 2220
gtgcacgaca tcggcaagaa cctggtcgac atcatcttga gcaacaacgg ctacgaagtg 2280
gtcaacatcg gcatcaagca gccaatcgcc accatcctcg aagtcgccga ggacaagagc 2340
gccgacgtgg tcggcatgtc gggcctgctg gtgaagtcga ccgtggtgat gaaggaaaac 2400
ctcgaggaga tgaacacccg gggagtcgcc gaaaagttcc cggtgctgct cggcggcgcg 2460
gcgttgacgc gcagctatgt cgaaaacgac ctggccgaga tctaccaggg cgaagtgcat 2520
tacgcgcgag acgctttcga gggcctgaag ttgatggaca ccatcatgag cgccaagcgc 2580
ggcgaggcgc ccgacgaaaa cagcccggaa gccattaagg cgcgtgagaa agaagccgaa 2640
cgtaaggccc gccaccagcg atccaaacgc attgccgcac agcgcaaagc cgccgaagaa 2700
ccagtcgagg tgcccgaacg ctccgatgtc gcggccgaca tcgaggtccc ggcgccgccg 2760
ttctggggtt cgcggatcgt caagggcctg gcggtggccg actacaccgg tctgctcgat 2820
gagcgcgcat tgtttttggg ccagtggggt ttacgcggcc agcgcggcgg tgagggtccg 2880
tcctacgaag atctcgtcga gaccgagggc cggccgcggc tgcggtactg gttggaccgg 2940
ctgtccaccg acggcatctt ggcgcacgcc gccgtggtgt acggctattt cccggcggtg 3000
tccgagggca acgacatcgt ggtgctcacc gagcccaagc ccgacgcccc ggtgcgctac 3060
cggtttcact tcccgcgcca gcagcgcggt cggtttttgt gcattgccga tttcatccgc 3120
tcgcgggagc tggccgccga gcgtggcgag gttgacgtgc tgccgttcca gctggtgacc 3180
atgggtcagc cgatcgcgga tttcgccaac gagctgttcg cgtccaacgc ctaccgcgac 3240
tacctggagg tgcacggtat cggcgtgcag ctcaccgagg cgctggccga gtactggcac 3300
cggcggatcc gtgaggagct caagttctcc ggggatcggg cgatggcggc cgaggatccg 3360
gaggcgaaag aagactattt caagctcggc taccgcggtg ctcgctttgc cttcggctac 3420
ggcgcatgcc cggatctgga ggaccgcgcc aagatgatgg cgctgctgga gcccgaacgc 3480
atcggtgtga cgttatccga ggaattacag ctgcatcccg aacagtcgac cgacgcgttc 3540
gtcctgcacc atccggaagc caagtacttc aacgtttaa 3579
<210> 2
<211> 90
<212> DNA
<213> rv3253c promoter region sequence
<400> 2
gcacccgtcc tctcgcactc gttgtgaccg cacagtactg ggtactctgc gaggatgacg 60
ggtcgcgtag ggaacccgaa ggaccacgcc 90
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 3
tgtcgtcgct gtacaccgca at 22
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 4
tgcacgatcg ccgaatccag c 21
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 5
aacgacatcg tggtgctcac c 21
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 6
ttggcttccg gatggtgcag ga 22
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 7
attccggcgc caatcaccac 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 8
ggccatcaac atgtgcaggt 20
<210> 9
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 9
gactaccaga agtgcctgga catcg 25
<210> 10
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 10
cacccggatg tgcagaccac acttg 25
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 11
aggcgctggc cgagtactgg ca 22
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 12
gcacccgtcc tctcgcactc 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 13
accgcacagt actgggtact 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 14
gaggatgacg ggtcgcgtag 20
Claims (10)
1. The mycobacterium tuberculosis drug resistance-related gene mutation site is characterized in that the gene mutation site is metH gene and/or rv3253c gene promoter region mutation site; the metH gene mutation site and its mutation type are A1108G, T1631C or A3292C with respect to the nucleic acid having the sequence of SEQ ID No. 1; relative to the nucleic acid with the sequence of SEQ ID No.2, the mutation site and the mutation type of the promoter region of the rv3253C gene are G7A, C31T, C33T, G35A, T39C, G40A, G42A or G57A.
2. Use of the gene mutation site according to claim 1 for preparing a reagent for detecting an aminosalicylic acid-resistant Mycobacterium tuberculosis, wherein the reagent comprises a primer composition and/or a probe for detecting the gene mutation site.
3. The use according to claim 2, wherein the primer composition comprises a primer having a nucleotide sequence of SEQ ID No.3 to 6 and/or a primer having a nucleotide sequence of SEQ ID No.7 to 8.
4. The use according to claim 2, wherein the probe has a sequence selected from the group consisting of SEQ ID Nos. 9 to 14 and the base complements thereof.
5. Use of the gene mutation site of claim 1 in the preparation of a kit for detecting aminosalicylic acid-resistant mycobacterium tuberculosis, wherein the kit comprises a primer composition and/or a probe for detecting the gene mutation site.
6. The use according to claim 5, wherein the primer composition comprises a primer having a nucleotide sequence of SEQ ID No.3 to 6 and/or a primer having a nucleotide sequence of SEQ ID No.7 to 8; the sequence of the probe is selected from SEQ ID No. 9-SEQ ID No.14 and the base complementary sequence thereof.
7. An kit for detecting an aminosalicylic acid-resistant mycobacterium tuberculosis, wherein the kit comprises a primer composition and/or a probe for detecting the mutation site of the gene according to claim 1.
8. The kit according to claim 7, wherein the primer composition comprises a primer having a nucleotide sequence of SEQ ID Nos. 3 to 6 and/or a primer having a nucleotide sequence of SEQ ID Nos. 7 to 8; the sequence of the probe is selected from SEQ ID No. 9-SEQ ID No.14 and the base complementary sequence thereof.
9. A rapid screening method for drug-resistant mutant base of Mycobacterium tuberculosis using the detection kit of any one of claims 7 to 8, wherein the rapid screening method is to amplify metH gene and its internal base sequence and detect the mutant site of the metH gene; and/or amplifying the rv3253c gene promoter region and detecting the rv3253c gene promoter region mutation site.
10. A second generation sequencing method for detecting the amino salicylic acid resistant mutant base of mycobacterium tuberculosis is characterized in that the method comprises the steps of carrying out high-throughput sequencing, comparing with a metH gene with a nucleotide sequence of SEQ ID No.1 and/or an rv3253c gene promoter region with a nucleotide sequence of SEQ ID No.2, and detecting whether base mutation occurs at 1108 th, 1631 th or 3292 th positions of the metH gene and the base sequence thereof and/or whether base mutation occurs at 7 th, 31 th, 33 th, 35 th, 39 th, 40 th, 42 th or 57 th positions of the rv3253c gene promoter region and the base sequence thereof.
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