Molecular marker for generation of drug resistance of mycobacterium tuberculosis, detection kit, and method

Abstract

The present invention relates to a molecular marker for generation of drug resistance of Mycobacterium tuberculosis and a detection kit and method therefor. The method comprises: detecting nonsynonymous mutation occurring at a specific genetic locus in a pncA gene, a rpsA gene or a panD gene, or detecting mutation occurring at a specific amino acid site in a pncA protein, a rpsA protein or a panD protein.

Description

Molecular markers, detection kits and methods for drug resistance of Mycobacterium tuberculosis Technical Field

[0001] The present invention relates to a molecular marker for drug resistance of Mycobacterium tuberculosis, a kit for detecting drug resistance of Mycobacterium tuberculosis and a detection method thereof, and in particular to a molecular marker for detecting Mycobacterium tuberculosis resistant to pyrazinamide (PZA), a kit and a detection method thereof. Background Art

[0002] Mycobacterium is a genus that includes various bacteria, among which Mycobacterium tuberculosis (MTB) is an acid-fast bacterium and the primary cause of pulmonary tuberculosis. Tuberculosis is a chronic infectious disease caused by MTB infecting the lungs, usually through droplets. Once MTB enters the lungs, they grow and multiply in the alveoli and cause an inflammatory response. Symptoms of pulmonary tuberculosis include cough, sputum, chest pain, fever, and fatigue. If left untreated, pulmonary tuberculosis can lead to serious complications such as lung infection, respiratory failure, tuberculoma, and tuberculous meningitis.

[0003] Currently, there are four most commonly used drugs for treating tuberculosis: isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB). PZA can inhibit the growth and reproduction of MTB by altering the pH level within MTB cells. Therefore, it is crucial in treating drug-resistant strains such as multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). However, the specific mechanism of action of PZA remains unclear.

[0004] However, some MTB have developed resistance to the above drugs, which may cause traditional treatment plans to fail because the strain may not be effectively killed by the therapeutic drugs. In order to detect which drugs MTB is resistant to, it is known that clinical samples (such as sputum) can be cultured on a culture medium containing antibiotics and the growth of MTB can be observed to determine its sensitivity to different drugs. However, the above method requires a lot of time and manpower costs, and its accuracy is also unsatisfactory.

[0005] In view of this, the inventors of the present invention have a deep understanding of the shortcomings and defects of the existing technology and are eager to improve and innovate it. After years of research, they have successfully developed the present invention, a molecular marker for drug resistance of Mycobacterium tuberculosis, a kit for detecting drug resistance of Mycobacterium tuberculosis, and a detection method thereof.

[0006] Summary of the Invention

[0007] To achieve the above-mentioned object, the present invention provides a molecular marker for drug resistance of Mycobacterium tuberculosis, which is any one of the following (A) to (K): (A) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence after the mutation is shown in SEQ ID NO: 2; (B) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 3, the nucleotide sequence after the mutation is shown in SEQ ID NO: 4; (C) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 5, the nucleotide sequence after the mutation is shown in SEQ ID NO: 6; (D) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 7, the nucleotide sequence after the mutation is shown in SEQ ID NO: 8; (E) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 9, the nucleotide sequence after the mutation is shown in SEQ ID NO: 10; (F) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 11, the nucleotide sequence after the mutation is shown in SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; (G) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 1 (H) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence after the mutation is shown in SEQ ID NO: 20; (I) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO: 21, and the nucleotide sequence after the mutation is shown in SEQ ID NO: 22; (J) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO: 23, and the nucleotide sequence after the mutation is shown in SEQ ID NO: 24; (K) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO: 25, and the nucleotide sequence after the mutation is shown in SEQ ID NO: 26.

[0008] The present invention also provides a molecular marker that causes tuberculosis to produce drug resistance, which is any one of the following (A') to (K'): (A') the 13th amino acid in the pncA protein is mutated from phenylalanine to tyrosine: Phe13Tyr; (B') the 68th amino acid in the pncA protein is mutated from tryptophan to leucine: Trp68Leu; (C') the 133rd amino acid in the pncA protein is mutated from isoleucine to leucine: Ile133Leu; (D') the 138th amino acid in the pncA protein is mutated from cysteine ​​to serine: Cys138Ser; (E') the pnc Amino acid 175 in the A protein mutated from methionine to lysine: Met175Lys; (F') amino acid 222 in the rpsA protein mutated from phenylalanine to leucine: Phe222Leu; (G') amino acid 225 in the rpsA protein mutated from phenylalanine to leucine: Phe225Leu; (H') amino acid 86 in the panD protein mutated from isoleucine to valine: Ile86Val; (I') amino acid 86 in the panD protein mutated from isoleucine to threonine: Ile86Thr; (J') amino acid 72 in the panD protein mutated from asparagine to threonine: Asn72Thr; (K') amino acid 90 in the panD protein mutated from tyrosine to aspartic acid: Tyr90Asp.

[0009] In one embodiment of the present invention, the drug to which Mycobacterium tuberculosis develops resistance is pyrazinamide (PZA), ethambutol (EMB), rifampicin (RIF), or isoniazid (INH).

[0010] The present invention further provides a kit for detecting drug resistance of Mycobacterium tuberculosis, which is used to detect the molecular markers mentioned above.

[0011] The present invention further provides a method for detecting drug resistance of Mycobacterium tuberculosis, which detects the molecular markers mentioned above.

[0012] In one embodiment of the present invention, the steps of the detection method include a method for detecting the gene sequence of MTB in a sample.

[0013] In one embodiment of the present invention, the method for detecting the gene sequence of MTB includes gene sequencing technology, which compares the sequencing results with the reference sequence to confirm the status of the molecular marker.

[0014] The present invention can quickly detect which drugs a sample is resistant to by detecting non-synonymous mutations at specific gene sites in the pncA gene, rpsA gene, or panD gene, or detecting mutations at specific amino acid sites in the pncA protein, rpsA protein, or panD protein, thereby helping doctors choose the most suitable tuberculosis treatment plan, improving the success rate of treatment, reducing the transmission and spread of tuberculosis, and thus protecting patients and public health. DETAILED DESCRIPTION

[0015] Definition of terms

[0016] This specification makes extensive use of many technical and scientific terms commonly used in the field of biotechnology. The following definitions are provided to ensure a clear and consistent understanding of this specification and claims, as well as the scope of these terms. Other terms not specifically defined below have the meanings commonly understood by those skilled in the art.

[0017] Unless otherwise indicated, the terms "or," "and," and "and" used in this specification refer to "or / and." Furthermore, the terms "include" and "comprising" are not restrictive open-ended conjunctions. The preceding paragraphs are merely systematic references and should not be construed as limiting the subject matter of the invention.

[0018] The term "tuberculosis bacteria" as used herein refers to all bacteria in the genus Mycobacterium that may cause pulmonary tuberculosis, including but not limited to Mycobacterium tuberculosis (MTB), Mycobacterium africanum, Mycobacterium microti, Mycobacterium avium, Mycobacterium fortuitum, or Mycobacterium bovis.

[0019] As used herein, "drug resistance" refers to the ability of bacteria, viruses, fungi, or other pathogens to resist one or more drugs. When treated with drugs, the pathogens may continue to grow and reproduce, leading to treatment failure or poor therapeutic efficacy. Mechanisms by which pathogens develop drug resistance include, but are not limited to: changes in the pathogen's structure due to changes in genotype or phenotype, thereby preventing the binding of the drug to the pathogen or reducing the affinity of the drug for the pathogen; genetic recombination in the pathogen, resulting in changes in the genetic composition and sequence that lead to drug resistance; pathogens acquiring exogenous genes, thereby acquiring drug resistance, etc.

[0020] The term "molecular marker" as used in this specification refers to a biological characteristic or property detected at the molecular level, which is used to identify organisms with specific characteristics or properties, including but not limited to: molecular markers at the gene level, molecular markers at the protein level, etc.

[0021] As used herein, a "non-synonymous mutation" refers to a change in the amino acid sequence of the protein encoded by the gene that occurs when one nucleotide in the gene sequence is replaced by another. A non-synonymous mutation can cause a change in the structure or function of the protein and may affect the phenotype of the organism.

[0022] As used herein, "sequence homology" refers to the degree of identity or similarity between two sequences as measured by an alignment of the sequences that maximizes the similarity between the aligned nucleotides, and is a function of the number of identical nucleotides, the number of total nucleotides, and the presence and length of gaps in the alignment. There are a variety of algorithms and computer programs available for determining sequence similarity using standard parameters. Preferably, sequence homology is measured using the BLASTn program for nucleic acid sequences, which is available through the National Center for Biological Information (www.ncbi.nlm.nih.gov / ) and described in, for example, Altschul et al. (1990), J Mol. Biol. 215: 403-410; Gish and States (1993), Nature Genet. 3: 266-272; Madden et al. (1996), Meth. Enzymol. 266: 131-141; Altschul et al. (1997), Nucleic Acids Res. 25: 3389-3402); Zhang et al. (2000), J. Comput. Biol. 7(1-2): 203-14.

[0023] Molecular markers at the gene level

[0024] The molecular markers for drug resistance of Mycobacterium tuberculosis provided by the present invention include molecular markers at the gene level, which are non-synonymous mutations occurring at specific gene sites in the pncA gene, rpsA gene, or panD gene, and include any one of the following molecular markers (A) to (K): (A) a non-synonymous mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide sequence after the mutation is shown in SEQ ID NO: 2; (B) a non-synonymous mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 3, and the nucleotide sequence after the mutation is shown in SEQ ID NO: 4; (C) a non-synonymous mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 5, and the nucleotide sequence after the mutation is shown in SEQ ID NO: 6; (D) a non-synonymous mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 7, and the nucleotide sequence after the mutation is shown in SEQ ID NO: 8; (E) a non-synonymous mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence after the mutation is shown in SEQ ID NO: 10; (F) a non-synonymous mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 11 NO:11, the nucleotide sequence after the mutation is shown in SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14; (G) a non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:15, the nucleotide sequence after the mutation is shown in SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18; (H) a non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:19, the nucleotide sequence after the mutation is shown in SEQ ID NO:20; (I) a non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:21, the nucleotide sequence after the mutation is shown in SEQ ID NO:22; (J) a non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:23, the nucleotide sequence after the mutation is shown in SEQ ID NO:24; (K) a non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:25, the nucleotide sequence after the mutation is shown in SEQ ID NO:26.

[0025] In one embodiment, the sequences of SEQ ID NOs: 1-26 mentioned in the molecular markers (A)-(K) provided herein further include sequences having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to these sequences, preferably sequences having sequence identity to the entire length (100%) of SEQ ID NOs: 1-26.

[0026] In one embodiment, the SEQ ID NOs: 1-26 mentioned in the molecular markers (A)-(K) provided by the present invention also include nucleotide sequences in which one or more nucleotides are substituted, deleted, or added but which can encode the same protein.

[0027] In one embodiment, the sequences of SEQ ID NOs: 1 to 26 mentioned in the molecular markers (A) to (K) provided herein further include sequences that differ from them by only 1 to 30 nucleotides, for example, by only 2, 3, 4, 5, 10, 15, 20, 25, or a number of nucleotides between any two of the foregoing values, without affecting the structure of the protein encoded therefrom.

[0028] Molecular markers at the protein level

[0029] The molecular markers for drug resistance of Mycobacterium tuberculosis provided by the present invention include molecular markers at the protein level, which are mutations at specific amino acid sites in the pncA protein, rpsA protein, or panD protein, and include any one of the following (A') to (K'): (A') the 13th amino acid in the pncA protein mutates from phenylalanine to tyrosine: Phe13Tyr; (B') the 68th amino acid in the pncA protein mutates from tryptophan to leucine: Trp68Leu; (C') the 133rd amino acid in the pncA protein mutates from isoleucine to leucine: Ile133Leu; (D') the 138th amino acid in the pncA protein mutates from cysteine ​​to serine: Cys138Ser; (E') the 175th amino acid in the pncA protein mutates from cysteine ​​to serine: Cys138Ser; The amino acid at position 222 in the (F') rpsA protein was mutated from phenylalanine to leucine: Phe222Leu; the amino acid at position 225 in the (G') rpsA protein was mutated from phenylalanine to leucine: Phe225Leu; the amino acid at position 86 in the (H') panD protein was mutated from isoleucine to valine: Ile86Val; the amino acid at position 86 in the (I') panD protein was mutated from isoleucine to threonine: Ile86Thr; the amino acid at position 72 in the (J') panD protein was mutated from asparagine to threonine: Asn72Thr; the amino acid at position 90 in the (K') panD protein was mutated from tyrosine to aspartic acid: Tyr90Asp.

[0030] Drug resistance

[0031] The molecular markers (A)-(K) and (A')-(K') provided by the present invention can be used to detect whether MTB is resistant to specific drugs, including but not limited to pyrazinamide (PZA), ethambutol (EMB), rifampicin (RIF) or isoniazid (INH), quinolones (QNS), capreomycin, kanamycin, amikacin, fluoroquinolones, linezolid, and the like. In this embodiment, the molecular markers can be used to detect whether MTB is resistant to PZA.

[0032] Detection kit

[0033] The kit for detecting drug resistance of Mycobacterium tuberculosis provided by the present invention can be used to detect molecular markers (A) to (K) and / or detect molecular markers (A') to (K'), and its contents are not limited. In one embodiment, the kit may include reagents used in PCR technology, such as primers of specific sequences, polymerase, reaction buffer, agarose gel for electrophoresis separation, etc. In another embodiment, the kit may include reagents used in DNA microarray, such as primers of multiple specific sequences, reaction buffer, etc. In another embodiment, the kit may include reagents used in whole genome sequencing or target region sequencing, such as probes used in sequencing reactions, etc.

[0034] Detection method

[0035] The method for detecting drug resistance of Mycobacterium tuberculosis provided by the present invention can be used to detect molecular markers (A) to (K) and / or detect molecular markers (A') to (K'), and its specific method steps are not limited. In one embodiment, the detection method based on PCR, including but not limited to amplification reaction, polymerase chain reaction, real-time PCR and other methods, can be used to detect the above-mentioned molecular markers to determine whether MTB is resistant to drugs. In another embodiment, the detection method based on DNA microarray technology can be used to detect MTB genomic DNA to simultaneously detect multiple genes and comprehensively determine whether MTB is resistant to drugs. In another embodiment, the detection method based on whole genome sequencing (NGS) can be used to perform high-throughput and high-efficiency sequencing of the whole genome of MTB using NGS technology, and detect the above-mentioned molecular markers by comparing with the reference genome sequence, so as to comprehensively determine whether MTB is resistant to drugs.

[0036] The molecular markers for drug resistance of Mycobacterium tuberculosis provided by the present invention are shown in Table 1 below:

[0037] Table 1

[0038] Several examples and comparative examples are listed below to illustrate the molecular markers for drug resistance in Mycobacterium tuberculosis, the kit for detecting drug resistance in Mycobacterium tuberculosis, and the detection method thereof. However, these examples are not intended to limit the present invention. Anyone skilled in the art will readily be able to make various modifications and improvements without departing from the spirit and scope of the present invention.

[0039] Example

[0040] 1. Drug susceptibility testing (DST)

[0041] DST is used to test the sensitivity or resistance of Mycobacterium tuberculosis to anti-tuberculosis drugs. This test uses a colorimetric assay to test the drug susceptibility of specific MTB isolates. The detailed process is as follows:

[0042] MTB were cultured in Lowenstein-Jensen medium to prepare an MTB suspension with a turbidity of 0.5 McFarland standard. After 24 hours of culture, 100 μL of PZA (8 mg / mL) was added to 2 mL of the MTB suspension. The reaction was then incubated at 37°C in the absence of CO2 for 4 days. 2 mL of the culture supernatant was mixed with 200 μL of 1 wt% ferrous ammonium sulfate and quantified at 490 nm using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). An absorbance value less than 0.1 was considered resistant to PZA.

[0043] The test results are shown in Table 2 below:

[0044] Table 2 Amino acid mutation status of MTB resistant to PZA. The numbers in brackets represent the number of samples n.

[0045] 2. Whole-genome sequencing of MTB genomic DNA

[0046] MTB gDNA was subjected to WGS using the ONT sequencing platform. The specific protocol was as follows: 0.5–1 μg of homogenized MTB gDNA was used for library preparation using the Ligation Sequencing Kit (SQK-LSK109; Oxford Nanopore Technologies (ONT), Oxford, UK) and the Native Barcoding Expansion Kit (EXP-NBD104 and 114; ONT) according to the manufacturer's instructions. Subsequently, 700 ng of the pooled library was placed in MinION flow cells (FLO-MIN106D R9.4.1; MinION MK1C device; ONT). Approximately 200,000 reads were generated from the barcoded DNA per isolate, achieving a sequencing depth of 200 reads.

[0047] 3. Detection of non-synonymous mutations in MTB genomic DNA

[0048] CLC Genomics Workbench (Qiagen v22.0.2; CLC bio, Denmark) was used to assess the quality and quantity of the raw reads. To assess the coverage of the raw reads against the MTB reference strain (M. tuberculosis H37Rv, NC_000962.3), the wf-alignment program in EPI2ME Labs software (ONT; v0.2.3) and CLC Genomics Workbench were used to assess the sequencing depth of the sequences. Non-synonymous mutations in MTB genomic DNA were assessed and detected by repeatedly using the wf-alignment program in EPI2ME Labs software (ONT; v0.2.9) and CLC Genomics Workbench (ONT; v0.2.3).

[0049] 4. Ligand-protein structure modeling and analysis

[0050] The protein structures of pncA (1SJ2), rpsA (4NNI), and panD (6OYY) were downloaded from the Protein Data Bank (PDBe, 14). The effects of the identified variants on the folding and stability of the proteins encoded by pncA, rpsA, or panD were simultaneously evaluated using three algorithms: mCSM-Stability, DUET, and SDM. In addition, the structures of PZA and its active form, POA, were downloaded from the chemical composition section of PDBe and applied to ligand-protein docking simulations using the Autodock vina website (https: / / vina.scripps.edu / ). The variant calling results generated using the CLC Genomics Workbench were applied to a 3D structure modeling pipeline that simulated the effects of the identified variants on the drug-target interactions of the present invention. The results are shown in Tables 3 to 5 below:

[0051] Table 3 Amino acid mutations of pcnA

[0052] [Table 4] Amino acid mutations in rpsA

[0053] Table 5 Amino acid mutations of panD

[0054] Based on the above results, the molecular markers for drug resistance of Mycobacterium tuberculosis, the kit for detecting drug resistance of Mycobacterium tuberculosis and the detection method thereof of the present invention, which include detecting non-synonymous mutations at specific gene sites in the pncA gene, rpsA gene, or panD gene, or detecting mutations at specific amino acid sites in the pncA protein, rpsA protein, or panD protein, can help doctors select the most suitable treatment plan for pulmonary tuberculosis, improve the success rate of treatment, reduce the transmission and spread of pulmonary tuberculosis, and thus protect patients and public health.

[0055] The embodiments described above are merely illustrative of the technical concepts and features of the present invention. Their purpose is to enable persons skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the patent scope of the present invention. In other words, any equivalent changes or modifications made in accordance with the spirit disclosed in the present invention should still be covered by the patent scope of the present invention.

Claims

1. A molecular marker for drug resistance of Mycobacterium tuberculosis, which is any one of the following (A) to (K): (A) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 1, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO: 2; (B) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO:3, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO:4; (C) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO:5, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO:6; (D) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO:7, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO:8; (E) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO:9, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO:10; (F) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO: 11, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; (G) a mutation occurring on the basis of the nucleotide sequence as shown in SEQ ID NO: 15, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18; (H) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO: 19, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO: 20; (I) a mutation occurring on the basis of the nucleotide sequence shown in SEQ ID NO:21, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO:22; (J) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO:23, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO:24; (K) A mutation occurring based on the nucleotide sequence shown in SEQ ID NO:25, wherein the nucleotide sequence after the mutation is shown in SEQ ID NO:

26.

2. A molecular marker for drug resistance of Mycobacterium tuberculosis, which is any one of the following (A') to (K'): (A') Amino acid 13 in the pncA protein mutated from phenylalanine to tyrosine: Phe13Tyr; (B') Amino acid 68 in the pncA protein was mutated from tryptophan to leucine: Trp68Leu; (C') Amino acid 133 in the pncA protein was mutated from isoleucine to leucine: Ile133Leu; (D') Amino acid 138 in the pncA protein was mutated from cysteine ​​to serine: Cys138Ser; (E') Amino acid 175 in the pncA protein was mutated from methionine to lysine: Met175Lys; (F') amino acid 222 in rpsA protein mutated from phenylalanine to leucine: Phe222Leu; (G') amino acid 225 in rpsA protein mutated from phenylalanine to leucine: Phe225Leu; (H') Amino acid 86 in panD protein mutated from isoleucine to valine: Ile86Val; (I') Amino acid 86 in panD protein mutated from isoleucine to threonine: Ile86Thr; (J') Amino acid 72 in panD protein mutated from asparagine to threonine: Asn72Thr; (K') The 90th amino acid in the panD protein mutated from tyrosine to aspartic acid: Tyr90Asp.

3. The molecular marker for drug resistance of Mycobacterium tuberculosis according to claim 1 or 2, wherein the drug to which the Mycobacterium tuberculosis develops resistance is pyrazinamide (PZA), ethambutol (EMB), rifampicin (RIF) or isoniazid (INH).

4. The molecular marker for drug resistance of Mycobacterium tuberculosis according to claim 1 or 2, wherein the drug to which the Mycobacterium tuberculosis produces resistance is pyrazinamide (PZA).

5. A kit for detecting drug resistance of Mycobacterium tuberculosis, used for detecting the molecular marker according to claim 1 or 2.

6. A method for detecting drug resistance of Mycobacterium tuberculosis, comprising detecting the molecular marker according to claim 1 or 2.

7. The method for detecting drug resistance of Mycobacterium tuberculosis according to claim 6, comprising the steps of detecting the gene sequence of MTB in a sample.

8. The method for detecting drug resistance of Mycobacterium tuberculosis according to claim 7, wherein the method for detecting the gene sequence of MTB comprises gene sequencing technology, which compares the sequencing result with the reference sequence to confirm the status of the molecular marker.

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