Fluoroquinolone resistance mutation sites and detection kits of Mycobacterium tuberculosis

Abstract

The invention provides a fluoroquinolone drug resistance mutation site of Mycobacterium tuberculosis and a detection kit. The drug resistance mutation sites are gyrA_A288D, gyrB_G436D and gyrB_D434E, wherein gyrA_A288D is a protein encoded by the gyrA gene, wherein the amino acid alanine at position 288 is replaced by aspartic acid; gyrB_G436D is a protein encoded by the gyrB gene, wherein the glycine at position 436 is replaced by aspartic acid; and gyrB_D434E is a protein encoded by the gyrB gene, wherein the aspartic acid at position 434 is replaced by glutamic acid. Whether any drug resistance mutation site of gyrA_A288D, gyrB_G436D and gyrB_D434E exists is detected, and the application is an application in a non-diagnosis or treatment field.

Description

Technical Field

[0001] The invention relates to the technical field of drug-resistant strains, and in particular to a Mycobacterium tuberculosis fluoroquinolone drug-resistant mutation site and a detection kit. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, which is mainly treated with anti-tuberculosis drugs. However, in actual practice, the number of patients who are detected with multidrug-resistant tuberculosis (MDR-TB) or tuberculosis resistant to a key anti-tuberculosis drug, rifampicin, and participate in the corresponding treatment program is far less than the actual number of patients estimated by epidemiological models. On the one hand, a considerable number of multidrug-resistant / rifampicin-resistant tuberculosis patients have not been discovered or have not received appropriate treatment; on the other hand, the number of these undetected and untreated patients is greater than expected, which may lead to the continued spread of the disease in the population and make efforts to control tuberculosis more difficult.

[0003] In order to enable drug-resistant patients to receive the correct diagnosis and start the most effective treatment as early as possible, the WHO End TB Strategy calls for universal drug susceptibility testing. Currently, the commonly used drug susceptibility tests in clinical practice include phenotypic drug susceptibility testing and genotypic testing. Among them, the phenotypic drug susceptibility testing of Mycobacterium tuberculosis usually takes more than a month to complete, requiring expensive and complex laboratory capabilities and high requirements for biosafety protection. Genotypic detection methods such as nucleic acid amplification testing (NAAT) and whole genome sequencing (WGS) with high specificity and sensitivity can help to make timely and accurate diagnosis of drug resistance in patients. In order to establish global standards for the prediction of genotypic drug resistance, the World Health Organization released the first version of the Mycobacterium tuberculosis resistance mutation catalog to guide genotypic testing. The catalog includes currently common anti-tuberculosis drugs, including first-line anti-tuberculosis drugs (rifampicin, isoniazid, ethambutol, pyrazinamide), and resistance mutation sites of second-line drugs in Group A (levofloxacin, moxifloxacin, bedaquiline, linezolid), Group B (clofazimine) and Group C (delamanide, amikacin, streptomycin, ethionamide, prothionamide), which greatly improves the limitations of traditional drug sensitivity testing, which is slow.

[0004] However, the drug-resistant mutation sites of tuberculosis vary from country to country. The drug-resistant mutation catalog of Mycobacterium tuberculosis currently issued shows that the drug-resistant sites that are strongly associated with drugs lack regional characteristics. At the same time, the accuracy and sensitivity of second-line drug detection are generally low. The false positive and false negative detection of drug-resistant strains caused by this will affect the further treatment of patients. The binding target of quinolones in Mycobacterium tuberculosis is DNA gyrase, which consists of two A subunits and two B subunits, encoded by gyrA and gyrB genes, respectively. Among them, codons 88 to 94 of gyrA and codons 500 to 538 of gyrB are quinolone resistance-determining regions, and non-synonymous mutations within them are identified as the main mechanism of fluoroquinolone resistance. When amino acid mutations occur in the gyrB gene, the three-dimensional structure of DNA gyrase will change. These structural changes may affect the binding site of fluoroquinolones and DNA gyrase, thereby reducing the drug's ability to inhibit the enzyme.

[0005] Fluoroquinolones have become commonly used clinical antibacterial drugs due to their broad antibacterial spectrum, strong antibacterial activity, low toxicity, wide distribution in the body, convenient administration, and no cross-resistance with other antibacterial drugs. However, due to their widespread use in the treatment of undiagnosed respiratory bacterial infections over the past 20 years, they have caused a high burden of quinolone resistance and increased the complexity of their resistance spectrum. At present, the quinolone resistance rate of Mycobacterium tuberculosis in China has gradually increased and the resistance level has gradually increased. Among them, the quinolone drug - moxifloxacin has become one of the key Group A drugs in the treatment of multidrug-resistant / drug-resistant tuberculosis. Therefore, further improving the detection rate and accuracy of molecular biological methods for rapid identification of quinolone resistance in Mycobacterium tuberculosis has guiding significance for clinical resistance detection and medication. Summary of the invention

[0006] The purpose of the present invention is to provide a fluoroquinolone resistance mutation site of Mycobacterium tuberculosis and a detection kit, by sequencing and screening clinical strains of Mycobacterium tuberculosis to obtain a new fluoroquinolone resistance mutation site of Mycobacterium tuberculosis, and design a detection kit based on the resistance mutation site to locate and detect the fluoroquinolone resistance of Mycobacterium tuberculosis.

[0007] To achieve the above objectives, in a first aspect, the technical scheme provides an application of a drug-resistant mutation site in detecting fluoroquinolone resistance of Mycobacterium tuberculosis, the drug-resistant mutation site being gyrA_A288D, gyrB_G436D or gyrB_D434E, wherein gyrA_A288D is a protein encoded by the gyrA gene, in which the amino acid alanine at position 288 is replaced by aspartic acid, gyrB_G436D is a protein encoded by the gyrB gene, in which the glycine at position 436 is replaced by aspartic acid, and gyrB_D434E is a protein encoded by the gyrB gene, in which the aspartic acid at position 434 is replaced by glutamic acid, and the presence of any one or a combination of two or more drug-resistant mutation sites of gyrA_A288D, gyrB_G436D and gyrB_D434E is detected, and the application is an application in a non-diagnostic or therapeutic field.

[0008] This protocol provides three drug-resistant mutation sites that can be used to predict tuberculosis resistance to fluoroquinolones. The three newly discovered drug-resistant mutation sites can serve non-diagnostic or therapeutic purposes in clinical practice: for example, they can provide important detection methods and monitoring indicators for disease control departments, help adjust and optimize drug resistance management strategies and anti-tuberculosis drug use guidelines, and slow down the development of drug resistance; they can provide important clues for the research and development of new anti-tuberculosis drugs, especially for existing drug-resistant pathogens, and help develop new drugs or drug combinations to overcome the limitations of existing treatments; they can help reveal the molecular mechanism of the development of Mycobacterium tuberculosis resistance, deepen researchers' understanding of the pathogenesis of tuberculosis, and provide a scientific basis for future treatment strategies.

[0009] The gyrA_A288D, gyrB_G436D and gyrB_D434E in this scheme were obtained by screening mutation sites strongly associated with drug resistance after antibiotic resistance testing of Mycobacterium tuberculosis. Specifically, the three mutation sites of gyrA_A288D, gyrB_G436D and gyrB_D434E are strongly associated with levofloxacin resistance. "gyrA_A288D" affects the structure of the adjacent loop on gyrB, restricting its movement. After the mutation, the energy required for the drug to escape from the target is significantly reduced, increasing the probability of drug off-target; while "gyrB_G436D, gyrB_D434E" may directly affect the drug binding site and also cause changes in its product conformation.

[0010] In a second aspect, the present scheme provides a primer combination for detecting fluoroquinolone-resistant mutation sites in Mycobacterium tuberculosis, comprising a gyrA amplification primer pair and a gyrB amplification primer pair, wherein the gyrA amplification primer pair comprises a gyrA forward primer having a primer sequence such as SEQ ID NO.1 and a gyrA backward primer having a primer sequence such as SEQ ID NO.2, and the gyrB amplification primer pair comprises a gyrB forward primer having a primer sequence such as SEQ ID NO.3 and a gyrB backward primer having a primer sequence such as SEQ ID NO.4.

[0011] The gyrA amplification primer pair in the primer combination provided in this scheme is used to amplify the full-length sequence of the gyrA gene, and the gyrB amplification primer pair is used to amplify the drug-resistant mutation region including the drug-resistant mutation sites of gyrA_A288D, gyrB_G436D and gyrB_D434E.

[0012] The primer sequence table of the primer combination is shown in Table 1 below:

[0013] Table 1 Primer sequence table of primer combination

[0014]

[0015] In some embodiments, the primer combination for detecting fluoroquinolone resistance mutation sites in Mycobacterium tuberculosis provided in this solution is used to amplify and sequence Mycobacterium tuberculosis to locate the resistance mutation sites. This solution can perform first-generation or second-generation sequencing to locate the resistance mutation sites.

[0016] Compared with the prior art, this technical solution has the following characteristics and beneficial effects:

[0017] By using an antibiotic tester on Mycobacterium tuberculosis, we screened out resistant strains that can still grow in the presence of fluoroquinolones. After screening and analyzing the resistant strains, we located three newly discovered resistance mutation sites of gyrA_A288D, gyrB_G436D and gyrB_D434E. We designed primer combinations for these three resistance mutation sites to achieve amplification and positioning, which can achieve rapid positioning of fluoroquinolone resistance sites in tuberculosis pathogens, allowing patients to obtain accurate resistance information earlier. DETAILED DESCRIPTION

[0018] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0019] Embodiment 1:

[0020] This protocol provides a method for screening for drug-resistant mutation sites of Mycobacterium tuberculosis fluoroquinolone resistance:

[0021] 1) Obtaining data sources: A total of 10,146 clinical strains of Mycobacterium tuberculosis from 72 counties across the country were collected, from which 9,079 clinical strains of Mycobacterium tuberculosis with phenotypic and genotypic data were selected as analysis data.

[0022] 2) Obtaining mutation sites: Whole genome sequencing of 9079 clinical strains of Mycobacterium tuberculosis was performed to obtain genomic data, and the "clockwork" mutation site screening scheme was used to identify the mutation sites in each sample, which can obtain the quality-controlled mutation site information in each sample. This information usually includes mutation type (such as SNP, insertion or deletion), location, frequency, etc.

[0023] 3) Drug resistance test: This study used three types of MYCOTB plates, namely "MYCOTBI", "UKMYC5" and "UKMYC6". These MYCOTB plates were pre-loaded with different antibiotic concentrations for drug susceptibility testing of 12 antibiotics (rifampicin RIF, isoniazid INH, ethambutol EMB, levofloxacin LFX, moxifloxacin MXF, amikacin AMI, kanamycin KAN, ethionamide ETO, cycloserine CFZ, linezolid LZD, daramicin DLM and bedaquiline BDQ). After 10-21 days of culture, two experimenters read the minimum inhibitory concentration (MIC) of each drug using the Vizion Digital viewing system, and inconsistent results were reread by a third experimenter. This program specifically selects strains that are resistant to levofloxacin LFX and fluoroquinolones. This is because the correlation between the drug-resistant mutation of this drug and the resistance type of the corresponding bacteria is not as good as that of first-line drugs. This program also screened several mutations associated with fluoroquinolones in the corresponding functional genes, which can improve the correlation between this mutation and the corresponding drug sensitivity results.

[0024] 4) Data statistics: According to the MIC value of the strain and the critical concentration of each drug, the strains were divided into resistant or susceptible, and the proportion of each mutation as a single mutation (PPS), positive predictive value (PPV) and its confidence interval (CIs), as well as odds ratio (ORs) and Fisher's exact test p value were calculated. PPS represents the proportion of a certain mutation appearing as a single mutation, that is, the proportion of strains containing only this mutation and no other resistance-related mutations; PPV refers to the probability that a strain with a specific mutation is actually resistant to the drug; the confidence interval provides a range to estimate the true value of PPV; ORs compare the probability that strains carrying a specific mutation are resistant to the drug compared to strains not carrying the mutation; this is a statistical test method used to determine whether the observed association is significant; and the Benjaminiand-Hochberg method is used to correct the obtained hypothesis test p value for multiple tests to obtain the q value, and the Wilcoxon rank sum test and Kruskal-Wallis test are used to quantify the relationship between mutations and MIC The Pearson chi-square test was used to find the correlation between geographical location and drug-resistant mutations.

[0025] Specifically, nonsynonymous mutations and insertion / deletion mutations (indels) were classified as either mutations that determined resistance (“R”) or mutations consistent with susceptibility (“S”). Synonymous mutations were assumed a priori to be neutral (“S”) and were masked unless they eliminated the start codon, in which case they were evaluated by the algorithm along with other types of mutations. Mutations in stop codons were not considered nonsynonymous mutations in this pipeline and were therefore not included in the analysis, but will be considered in the next version of the mutation catalog. Any nonsynonymous mutation or indel mutation that was identified as the only such mutation in an isolate that was phenotypically resistant (i.e., a solitary mutation) was classified as “R”. Any nonsynonymous mutation or indel mutation that was only present in a phenotypically susceptible isolate or was always associated with a susceptible phenotype when it was the only nonsynonymous mutation or indel mutation was classified by the algorithm as a mutation consistent with “susceptibility” (“algorithm S” or “aS”). Any mutation that could not be classified as “R” or “aS,” that had never occurred as a single mutation and that had never occurred in a phenotypically susceptible strain, was classified as undefined (“U”).

[0026] The numbers of resistant and susceptible isolates with and without mutations were summarized in a 2 x 2 contingency table, and the odds ratios (ORs) were calculated by Fisher's exact test, along with the corresponding confidence intervals (CIs) and P values, which were calculated based on the hypergeometric distribution. To control for multiple testing, the Benjamini-Hochberg correction procedure was used, with a false discovery rate (FDR) set at 5%. This correction procedure was applied to the number of tests for R, aS, and U mutations identified by the algorithm for a given drug, as well as to the number of mutations identified as neutral by the algorithm in a preliminary step. Because these neutral mutations were excluded before the algorithm was run, the number of tests used for correction did not include mutations that were labeled as neutral based solely on the study by Merker et al. (8). The same statistical procedure was also applied to the odds ratio of a single mutation (OR SOLO), in which case only the number of isolates with a single mutation was counted and compared to the corresponding number of isolates without the mutation: For all mutations, PPV|SOLO was calculated, which is the number of times a mutation was observed as a single mutation in the presence of a resistant phenotype, and the association between the mutation and MIC was quantified using the Wilcoxon rank sum test and the Kruskal-Wallis test, divided by the sum of this number and the number of times the mutation was observed in the presence of a susceptible phenotype; 95% confidence intervals were obtained using the Clopper-Pearson method. These statistical indices were then used to stratify and prioritize mutations using the confidence grading method described below, and the algorithmic labels "R", "aS", "S", and "U" were replaced according to the grading criteria.

[0027] The matched and collated phenotypic and genotypic data were then fed into an algorithm to identify "single" mutations, i.e., single mutations in a set of genes of interest that best explain the observed resistance phenotype after excluding neutral mutations (not associated with resistance). The final data for all mutations and phenotypes were then statistically evaluated for confidence grading. This included determining the odds ratio (OR) and positive predictive value (PPV) for the association of a mutation with resistance. Due to the large and heterogeneous data set, strict criteria were applied and the boundaries of the 95% confidence interval (CI) were used when applicable.

[0028] Each mutation was classified as "associated with drug resistance," "not associated with drug resistance," or "of uncertain significance." In addition, intermediate categories of "associated with drug resistance" and "not associated with drug resistance" were included: "associated with drug resistance but with weak evidence" and "not associated with drug resistance but with weak evidence," for a total of five groups.

[0029] 5) Resistance site classification: According to the OR, PPV, and FDRq values ​​of the mutation, the mutations are divided into 5 groups: 1. Strongly associated with resistance; 2. Associated with resistance but with weak evidence; 3. Uncertain significance; 4. Unrelated to resistance but with weak evidence; 5. Unrelated to resistance. Specifically, if the mutation occurs in more than five samples, the 95% CI lower limit of the PPV is greater than or equal to 0.25, the OR is greater than 1, and the FDR-corrected p value is less than 0.05, it is classified into Group 1 or Group 2, among which mutations that appear in the WHO catalog are classified as Group 1, and the rest are classified as Group 2; mutations that do not meet the above conditions are classified as Group 3, among which mutations that appear in Group 1 and Group 2 of the WHO catalog are promoted to Group 2, and any non-synonymous mutations in the RIF resistance-determining region of rpoB and any premature termination, insertion, or deletion in ethA or katG are classified as Group 2.

[0030] 6) Data statistics: A total of 11,065 gene mutations were identified in the data, of which 202 (1.83%) were classified as associated with phenotypic resistance (Group 1 or Group 2). The newly discovered mutations in the data set are "gyrA_A288D", "gyrB_G436D", and "gyrB_D434E", which are strongly associated with levofloxacin resistance (Group 1). "gyrA_A288D" affects the structure of the adjacent gyrB loop, limiting its movement. After the mutation, the energy required for the drug to leave the target is significantly reduced, increasing the probability of drug off-target. "gyrB_G436D, gyrB_D434E" may directly affect the drug binding site and also cause conformational changes in its product.

[0031] Embodiment 2:

[0032] This protocol provides a design of a primer combination for detecting fluoroquinolone resistance mutation sites in Mycobacterium tuberculosis:

[0033] The application team downloaded a data set containing 19,903 Mycobacterium gyrA and 304 gyrB complete gene sequences from the PubMLST database. According to the length of the gyrA and gyrB genes in the H37Rv genome, the fragments with a length of 1400-1600 bp of the gyrA gene and 2000-2100 bp of the gyrB gene in the downloaded data were retained as complete gene sequences for subsequent analysis. After using cd-hit to remove redundant sequences with a similarity of more than 99%, the sequencing data was aligned using MAFFT for primer design. Finally, the application team designed two pairs of amplification primers, including a gyrA amplification primer pair and a gyrB amplification primer pair, wherein the gyrA amplification primer pair includes a gyrA forward primer with a primer sequence such as SEQIDNO.1 and a gyrA backward primer with a primer sequence such as SEQIDNO.2, and the gyrB amplification primer pair includes a gyrB forward primer with a primer sequence such as SEQIDNO.3 and a gyrB backward primer with a primer sequence such as SEQIDNO.4. These two pairs of amplification primers are suitable for amplifying the full-length sequence and common drug-resistant mutation regions of the gyrA and gyrB genes, and perform well in both third-generation and first-generation sequencing technologies.

[0034] The gyrA amplification primer pair and the gyrB amplification primer pair designed by the application team can cover almost all tuberculosis samples. Secondly, the previous second-generation primers for gyrA and gyrB require multiple PCRs, which may cause false negatives due to primer off-target due to amplification efficiency. As the first-generation sequencing is prolonged, the accuracy of many bases cannot be guaranteed. This solution adjusts the amplification segments of the gyrA amplification primer pair and the gyrB amplification primer pair according to the location of the corresponding drug-resistant mutation site, which can ensure that the region can be amplified by a pair of primers while also ensuring the accuracy of identifying mutations.

[0035] Embodiment three:

[0036] This scheme provides an application of a primer combination for detecting fluoroquinolone resistance mutation sites in Mycobacterium tuberculosis. The application team randomly selected 15 samples infected with Mycobacterium tuberculosis from samples from 2013 to 2018 for first-generation and second-generation PCR sequencing. The 15 samples infected with Mycobacterium tuberculosis included gyrA and gyrB, of which the first 13 strains were amplified with the gyrA amplification primer pair, and the last 2 strains were amplified with the gyrB amplification primer pair.

[0037] First-generation sequencing of 17 strains of Mycobacterium tuberculosis: Genomic DNA of Mycobacterium tuberculosis was extracted using a commercial DNA extraction kit or standard laboratory method. The extracted DNA was tested for quality and concentration to ensure that it was suitable for subsequent PCR amplification. Next, the extracted DNA was used as a template to PCR amplify the gyrA and gyrB genes using the gyrA amplification primer pair and gyrB amplification primer pair of this protocol. After amplification, the quality and size of the PCR product were verified by agarose gel electrophoresis to ensure that the expected fragment was successfully amplified. Subsequently, the PCR amplification product was purified to remove primer dimers, unused dNTPs, and other impurities. The purified PCR product can be directly used for Sanger sequencing. The purified PCR product was mixed with sequencing primers and sequencing reagents for Sanger sequencing reaction. After the reaction, capillary electrophoresis was performed using a sequencer to read the fluorescently labeled termination nucleotides and generate a sequencing map.

[0038] Perform next-generation sequencing: Extract genomic DNA from Mycobacterium tuberculosis to ensure that the extracted DNA has high purity and integrity. The quality and concentration of the DNA are evaluated by agarose gel electrophoresis and spectrophotometer. In order to perform Illumina sequencing, the extracted genomic DNA needs to be constructed into a library. Fragment the DNA and use ultrasound to interrupt it, and randomly shear the genomic DNA into sequencing fragments with a length of 200-500 bp. The interrupted DNA fragments are end-repaired and A tails and adapters are added. After the library is constructed, PCR is used to amplify the library to increase the amount of DNA required for sequencing. The quality of the library is tested by agarose gel electrophoresis or fragment analyzer. After the library is qualified, the next-generation sequencing is performed on the Myseq sequencing platform PE150.

[0039] The sequencing results of the first-generation and second-generation sequencing are shown in Table 2 below, where the last two columns in Table 2 (reference base and mutant base) correspond to the second-generation sequencing results. The values ​​outside the brackets mentioned at the genome mutation position in the table are the nucleotide mutation positions in the entire genome.

[0040] The first-generation sequencing results showed that among the 15 samples, 2 strains were detected with the "gyrA_A288D" mutation, 2 strains were homozygous mutations, 1 strain was detected with the "gyrB_G436D" mutation, and 1 strain was detected with the "gyrB_D434E".

[0041] The second-generation sequencing results showed that the gyrA_A288D mutation was detected in a total of four samples. In addition to the sample detected by the first-generation sequencing, the other two samples showed heterozygous mutations at this site.

[0042] Table 2 Sequencing results of samples

[0043] Sample name Genomic mutation location (within the gyrA gene) First-generation sequencing results (amino acid changes) Reference base (frequency %) Mutated bases (frequency) 031 8164（863） CA(AD) A(1%) C(99%) 1159 8164（863） - 1394 8164（863） - 1395 8164（863） CA(AD) A(0%) C(100%) 1513 8164（863） - 1574 8164（863） - 1576 8164（863） - 247 8164（863） - A(13%) C(87%) 288 8164（863） - 356 8164（863） - A(40%) C(60%) 5 8164（863） - 776 8164（863） - 922 8164（863） - 293 6546(1307) GA(GD) G(0%) A(100%) 009 6541(1302) CA(DE) C(1%) A(99%)

[0044] It should be noted that 1302 CA in sample 293 and 1307 GA in sample 009 are both nucleotide mutations, which are counted as [P1] for gryB first-generation sequencing. Since the counting starts from 0, the actual mutation position is increased by 1 at the corresponding position, and the corresponding amino acid mutations are gyrB_G436D and gyrB_D434E1.

[0045] Different analysis results show that compared with traditional melting curves and first-generation sequencing methods, the use of high-throughput sequencing technology (second-generation or third-generation sequencing) can improve the detection accuracy of heterozygous drug-resistant sites.

[0046] In addition, this protocol conducted subsequent drug sensitivity tests on strains containing three sites, "gyrA_A288D", "gyrB_G436D", and "gyrB_D434E". The results of the fluoroquinolone drug sensitivity test showed that the median minimum inhibitory concentration (MIC) of the gyrA_A288D mutant strain was 2, while the median MIC of the gyrB_G436D and gyrB_D434E mutant strains was 1. Compared with the control group, the MICs of these mutant strains increased, indicating that these independent mutations can lead to a significant increase in the resistance of the strains to fluoroquinolones. These findings provide important experimental data for understanding the resistance mechanisms of fluoroquinolones in different strains, and also provide a theoretical basis for the development of new antibacterial strategies.

[0047] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims

1. An application of a drug-resistant mutation site in detecting fluoroquinolone resistance in Mycobacterium tuberculosis, characterized in that: The fluoroquinolone drug for Mycobacterium tuberculosis is levofloxacin, and the drug-resistant mutation site is gyrB_G436D, wherein gyrB_G436D is the replacement of glycine at position 436 on the protein encoded by the gyrB gene by aspartic acid, and the presence of the drug-resistant mutation site of gyrB_G436D is detected. The application is an application in non-diagnostic or therapeutic fields.

2. An application of a drug-resistant mutation site in detecting fluoroquinolone resistance in Mycobacterium tuberculosis, characterized in that: The fluoroquinolone drug for Mycobacterium tuberculosis is levofloxacin, and the drug-resistant mutation sites are gyrB_G436D and gyrB_D434E, wherein gyrB_G436D is the replacement of glycine at position 436 on the protein encoded by the gyrB gene by aspartic acid, and gyrB_D434E is the replacement of aspartic acid at position 434 on the protein encoded by the gyrB gene by glutamic acid, and the presence of drug-resistant mutation sites of gyrB_G436D and gyrB_D434E is detected, and the application is an application in non-diagnostic or therapeutic fields.

3. Use of the drug-resistant mutation site according to any one of claims 1 or 2 in detecting fluoroquinolone resistance in Mycobacterium tuberculosis, characterized in that: The drug-resistant mutation sites are obtained by screening mutation sites that are strongly associated with drug resistance after conducting antibiotic resistance testing on Mycobacterium tuberculosis.

4. Use of the drug-resistant mutation site according to any one of claims 1 or 2 in detecting fluoroquinolone resistance in Mycobacterium tuberculosis, characterized in that: The drug-resistant mutation sites were strongly correlated with levofloxacin resistance.

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