A molecular marker for drug resistance in Mycobacterium tuberculosis, a reagent kit for detecting drug resistance in Mycobacterium tuberculosis, and a detection method thereof.

Abstract

A molecular marker for drug resistance in Mycobacterium tuberculosis, a reagent kit for detecting drug resistance in Mycobacterium tuberculosis, and a detection method thereof, comprising detecting nonsynonymous mutations at specific gene loci in the pncA gene, rpsA gene, or panD gene, or detecting mutations at specific amino acid loci 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 infection and spread of pulmonary tuberculosis, thereby protecting patients and public health.

Description

Technical Field

[0001] This invention relates to a molecular marker for drug resistance in Mycobacterium tuberculosis, a reagent kit for detecting drug resistance in Mycobacterium tuberculosis, and a detection method thereof, particularly to a molecular marker, a reagent kit, and a detection method thereof for detecting Mycobacterium tuberculosis resistant to pyrazinamide (PZA). Prior Technology

[0002] Mycobacterium is a genus of bacteria that includes many species, among which Mycobacterium tuberculosis (MTB) is an acid-fast bacterium and the main cause of pulmonary tuberculosis. Pulmonary tuberculosis is a chronic infectious disease caused by MTB infection of the lungs, usually transmitted through droplets. Once in the lungs, MTB grows and multiplies in the alveoli, causing an inflammatory response in the lungs. Symptoms of pulmonary tuberculosis include cough, sputum production, chest pain, fever, and fatigue. If left untreated, pulmonary tuberculosis can lead to serious complications such as lung infections, respiratory failure, tuberculoma, and tuberculous meningitis.

[0003] Currently, there are four commonly used drugs for treating tuberculosis: isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB). PZA, in particular, inhibits the growth and reproduction of MTB cells by altering their pH, making it crucial for 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 strains have developed resistance to these drugs, which may cause traditional treatment regimens to fail because the strain may not be effectively killed by the treatment drugs. To detect which drugs MTBs are resistant to, it is known that clinical samples (such as sputum) can be cultured on a medium containing antibiotics and the growth of MTBs can be observed to determine their sensitivity to different drugs. However, the above method requires a lot of time and manpower, and its accuracy is also unsatisfactory.

[0005] In view of this, the inventor of this case deeply understands the shortcomings and defects of the previous case, and is eager to improve and innovate. After years of research, the inventor has successfully developed a molecular marker for drug resistance in Mycobacterium tuberculosis, a reagent kit for detecting drug resistance in Mycobacterium tuberculosis, and a detection method thereof. Summary of the Invention

[0006] To achieve the above objectives, the present invention provides a molecular marker for inducing drug resistance in Mycobacterium tuberculosis, which is any one of the following (A) to (K): (A) a mutation based on the nucleotide sequence shown in SEQ ID NO:1, the mutated nucleotide sequence being shown in SEQ ID NO:2; (B) a mutation based on the nucleotide sequence shown in SEQ ID NO:3, the mutated nucleotide sequence being shown in SEQ ID NO:4; (C) a mutation based on the nucleotide sequence shown in SEQ ID NO:5, the mutated nucleotide sequence being shown in SEQ ID NO:6; (D) a mutation based on the nucleotide sequence shown in SEQ ID NO:7, the mutated nucleotide sequence being shown in SEQ ID NO:8; (E) a mutation based on the nucleotide sequence shown in SEQ ID NO:9, the mutated nucleotide sequence being shown in SEQ ID NO:10; (F) a mutation based on the nucleotide sequence shown in SEQ ID NO:11, the mutated nucleotide sequence being shown in SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14; (G) a mutation based on the nucleotide sequence shown in SEQ ID NO:11, the mutated nucleotide sequence being shown in SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14; (H) A mutation occurring based on the nucleotide sequence shown in SEQ ID NO:15, resulting in the nucleotide sequence shown in SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18; (I) A mutation occurring based on the nucleotide sequence shown in SEQ ID NO:19, resulting in the nucleotide sequence shown in SEQ ID NO:20; (I) A mutation occurring based on the nucleotide sequence shown in SEQ ID NO:21, resulting in the nucleotide sequence shown in SEQ ID NO:22; (J) A mutation occurring based on the nucleotide sequence shown in SEQ ID NO:23, resulting in the nucleotide sequence shown in SEQ ID NO:24; (K) A mutation occurring based on the nucleotide sequence shown in SEQ ID NO:25, resulting in the nucleotide sequence shown in SEQ ID NO:26.

[0007] This invention also provides a molecular marker for inducing drug resistance in Mycobacterium tuberculosis, which is any one of the following (A')~(K'): (A') the 13th amino acid in the pncA protein, mutated from phenylalanine to tyrosine: Phe13Tyr; (B') the 68th amino acid in the pncA protein, mutated from tryptophan to leucine: Trp68Leu; (C') the 133rd amino acid in the pncA protein, mutated from isoleucine to leucine: Ile133Leu; (D') the 138th amino acid in the pncA protein, mutated from cysteine ​​to serine: Cys138Ser; (E') the 175th amino acid in the pncA protein, mutated from methionine to lysine: Met175Lys. (F') The 222nd amino acid in the rpsA protein is mutated from phenylalanine to leucine: Phe222Leu; (G') The 225th amino acid in the rpsA protein is mutated from phenylalanine to leucine: Phe225Leu; (H') The 86th amino acid in the panD protein is mutated from isoleucine to valine: Ile86Val; (I') The 86th amino acid in the panD protein is mutated from isoleucine to sine: Ile86Thr; (J') The 72nd amino acid in the panD protein is mutated from aspartic acid to sine: Asn72Thr; (K') The 90th amino acid in the panD protein is mutated from tyrosine to aspartic acid: Tyr90Asp.

[0008] In one embodiment of the present invention, the drug that induces resistance in Mycobacterium tuberculosis is pyrazinamide (PZA).

[0009] The present invention also provides a reagent kit for detecting drug resistance in Mycobacterium tuberculosis, for detecting molecular markers as described above.

[0010] The present invention also provides a method for detecting drug resistance in Mycobacterium tuberculosis, which detects the molecular markers described above.

[0011] In one embodiment of the present invention, the detection method includes a method for detecting the gene sequence of MTB in a sample.

[0012] In one embodiment of the present invention, the method for detecting the gene sequence of MTB includes gene sequencing technology, which involves comparing the sequencing results with a reference sequence to confirm the status of the molecular marker.

[0013] This invention can rapidly detect drug resistance to pyrazinamide (PZA) by detecting nonsynonymous mutations at specific gene sites in the pncA, rpsA, or panD genes, or mutations at specific amino acid sites in the pncA, rpsA, or panD proteins. This helps doctors select the most suitable treatment plan for tuberculosis, improves treatment success rates, reduces the transmission and spread of tuberculosis, and thus protects patients and public health. Implementation

[0014] [Terminology Definition]

[0015] This specification extensively uses many technical and scientific terms commonly used in the field of biotechnology. In the following description, to provide a clear and consistent understanding of the scope of this specification and the claims, as well as the scope to which these terms are applied, the following definitions are provided. Other terms not specifically defined below have meanings commonly understood by those skilled in the art.

[0016] The words "or," "and," and "and" used in this specification, unless otherwise stated, refer to "or / and." Furthermore, the terms "comprising" and "including" are not restrictive open-ended conjunctions. The foregoing paragraphs are for systematic reference only and should not be construed as limiting the subject of the invention.

[0017] The term "tuberculosis bacillus" as used in this instruction manual 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, etc.

[0018] The term "drug resistance" as used in this instruction manual refers to the ability of bacteria, viruses, fungi, or other pathogens to resist one or more drugs. When an individual uses a drug for treatment, the pathogen may continue to grow and multiply, leading to treatment failure or poor treatment efficacy. The mechanisms by which pathogens develop drug resistance include, but are not limited to: changes in the structure of the pathogen due to changes in genotype or phenotype, thereby preventing the drug from binding to the pathogen or reducing the affinity of the drug for the pathogen; gene recombination in the pathogen, resulting in changes in gene composition and sequence that lead to the development of drug resistance; and the pathogen acquiring exogenous genes, thereby acquiring drug resistance.

[0019] The term "molecular marker" as used in this specification refers to a biological characteristic or property detected at the molecular level, 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.

[0020] The term "non-synonymous mutation" as used in this manual refers to a change in the amino acid sequence of a protein encoded by a gene when one nucleotide in the gene sequence is replaced by another nucleotide. Non-synonymous mutations can cause changes in the structure or function of a protein, which may affect the phenotype of an organism.

[0021] The term "sequence homology" as used in this specification refers to the degree of identity or similarity between two sequences as measured by sequence alignment (which maximizes the similarity between aligned nucleotides), and is a function of the number of identical nucleotides, the total number of nucleotides, and the presence and length of gaps in the sequence alignment. Various algorithms and computer programs can be used to determine sequence similarity using standard parameters. Preferably, sequence homology is measured using the BLASTn program for nucleic acid sequences, which is available from the National Center for Biotechnology 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. 25:33 89-3402; Zhang et al. (2000), J. Comput. Biol. 7(I-2):203-14.

[0022] [Molecular markers at the gene level]

[0023] The molecular markers for drug resistance in Mycobacterium tuberculosis provided by this invention are gene-level molecular markers, which are non-synonymous mutations at specific gene loci 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 based on the nucleotide sequence shown in SEQ ID NO:1, with the mutated nucleotide sequence shown in SEQ ID NO:2; (B) a non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:3, with the mutated nucleotide sequence shown in SEQ ID NO:4; (C) a non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:5, with the mutated nucleotide sequence shown in SEQ ID NO:6; (D) a non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:7, with the mutated nucleotide sequence shown in SEQ ID NO:8; (E) a non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:9, with the mutated nucleotide sequence shown in SEQ ID NO:10; (F) a non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:9; (G) A non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:11, resulting in the nucleotide sequence 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, resulting in the nucleotide sequence 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, resulting in the nucleotide sequence shown in SEQ ID NO:20; (I) A non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:21, resulting in the nucleotide sequence shown in SEQ ID NO:22; (J) A non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:23, resulting in the nucleotide sequence shown in SEQ ID NO:24; (K) A non-synonymous mutation based on the nucleotide sequence shown in SEQ ID NO:25, resulting in the nucleotide sequence shown in SEQ ID NO:26.

[0024] In one embodiment, the sequences SEQ ID NO:1-26 mentioned in the molecular markers (A)-(K) provided by the present invention 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 homology with those sequences; preferably, sequences having homology with the entire length (100%) of the sequences SEQ ID NO:1-26.

[0025] In one embodiment, the sequences SEQ ID NO:1-26 mentioned in the molecular markers (A) to (K) provided by the present invention further include nucleotide sequences that have been substituted, deleted, or added with one or more nucleotides, but can encode the same protein.

[0026] In one embodiment, the sequences SEQ ID NO: 1 to 26 mentioned in the molecular markers (A) to (K) provided by the present invention, without affecting the structure of the protein they encode, further include sequences that differ from them by only 1 to 30 nucleotides. For example, these sequences can be sequences that differ from them by only 2, 3, 4, 5, 10, 15, 20, 25, or any number of nucleotides between the aforementioned two values.

[0027] [Molecular markers at the protein level]

[0028] The molecular markers for drug resistance in Mycobacterium tuberculosis provided by this invention are molecular markers at the protein level, which are mutations at specific amino acid sites in pncA, rpsA, or panD proteins, including any one of the following (A') to (K'): (A') Amino acid at position 13 of pncA protein, mutated from phenylalanine to tyrosine: Phe13Tyr; (B') Amino acid at position 68 of pncA protein, mutated from tryptophan to leucine: Trp68Leu; (C') Amino acid at position 133 of pncA protein, mutated from isoleucine to leucine: Ile133Leu; (D') Amino acid at position 138 of pncA protein, mutated from cysteine ​​to serine: Cys138Ser; (E') Amino acid at position 175 of pncA protein... The amino acids at position 222 of the rpsA protein, phenylalanine, are mutated to leucine: Met175Lys; the amino acid at position 222 of the rpsA protein, phenylalanine, is mutated to leucine: Phe222Leu; the amino acid at position 225 of the rpsA protein, phenylalanine, is mutated to leucine: Phe225Leu; the amino acid at position 86 of the panD protein, isoleucine, is mutated to valine: Ile86Val; the amino acid at position 86 of the panD protein, isoleucine, is mutated to sine: Ile86Thr; the amino acid at position 72 of the panD protein, aspartic acid, is mutated to sine: Asn72Thr; and the amino acid at position 90 of the panD protein, tyrosine, is mutated to aspartic acid: Tyr90Asp.

[0029] [Drug-resistant drugs]

[0030] The molecular markers (A)~(K) and (A')~(K') provided by this invention can be used to detect whether MTB is resistant to a specific drug, including but not limited to pyrazinamide (PZA), ethambutol (EMB), ripanmycin (RIF) or isoniazid (INH), quinolones (QNS), capreomycin, kanamycin, amikacin, fluoroquinolones, linezolid, etc. In this embodiment, the above molecular markers can be used to detect whether MTB is resistant to PZA.

[0031] [Testing Reagent Set]

[0032] The reagent kit for detecting drug resistance in Mycobacterium tuberculosis provided by this invention can be used to detect molecular markers (A)~(K) and / or molecular markers (A')~(K'), and its contents are not limited. In one embodiment, the reagent kit may contain reagents used in PCR technology, such as primers for specific sequences, polymerases, reaction buffers, agarose gels for electrophoretic separation, etc. In another embodiment, the reagent kit may contain reagents used in DNA microarrays, such as primers for multiple specific sequences, reaction buffers, etc. In yet another embodiment, the reagent kit may contain reagents used in whole-genome sequencing or target region sequencing, such as probes used in sequencing reactions, etc.

[0033] [Detection Method]

[0034] The method for detecting drug resistance in Mycobacterium tuberculosis provided by this invention can be used to detect molecular markers (A)~(K) and / or molecular markers (A')~(K'), and the specific method steps are not limited. In one embodiment, a PCR-based detection method, including but not limited to amplification reaction, polymerase chain reaction, real-time PCR, etc., can be used to detect the above-mentioned molecular markers to determine whether MTB has developed drug resistance. In another embodiment, a DNA microarray technology-based detection method can be used to detect multiple genes simultaneously by detecting MTB genomic DNA to comprehensively determine whether MTB has developed drug resistance. In yet another embodiment, a next-generation sequencing (NGS)-based detection method can be used to perform high-volume and high-efficiency sequencing of the entire MTB genome using NGS technology, and the status of the above-mentioned molecular markers can be detected by comparing with a reference genome sequence, thereby comprehensively determining whether MTB has developed drug resistance.

[0035] The molecular markers for drug resistance in Mycobacterium tuberculosis provided by this invention are shown in Table 1 below:

[0036] The following examples and comparative examples illustrate the molecular markers for drug resistance in Mycobacterium tuberculosis, the reagent kits for detecting drug resistance in Mycobacterium tuberculosis, and the detection methods thereof. However, these are not intended to limit the present invention. Any person skilled in the art may make various modifications and refinements without departing from the spirit and scope of the present invention.

[0037] [Example]

[0038] 1. Drug susceptibility testing (DST)

[0039] The DST (Disseminated Substance Testing) is used to test the susceptibility or resistance of Mycobacterium tuberculosis (MTB) to anti-tuberculosis drugs. This test uses a colorimetric assay to determine the drug susceptibility of specific MTB isolates. The detailed procedure is as follows: MTB is 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) is added to 2 mL of the MTB suspension, and the mixture is then incubated at 37°C under carbon dioxide-free conditions for 4 days. 2 mL of the culture supernatant is mixed with 200 μL of ferrous ammonium sulfate (1 wt%), and quantification is performed at 490 nm using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). A absorbance value less than 0.1 indicates resistance to PZA.

[0040] The test results are shown in Table 2 below:

[0041] 2. Perform whole-genome sequencing on the genomic DNA of MTB.

[0042] MTB gDNA was sequenced using the ONT platform for WGS. The specific procedure is as follows: 0.5–1 μg of homogenized MTB gDNA was prepared into a library 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 instruction manual. Then, 700 ng of the pooled library was placed in MinION flow cells (FLO-MIN106D R9.4.1; MinION MK1C device; ONT). Approximately 200,000 barcode DNA readings were generated for each isolate, achieving a sequencing depth of 200 μL.

[0043] 3. Detection of non-synonymous mutations in MTB genomic DNA

[0044] The quality and quantity of the raw reads were evaluated using CLC Genomics Workbench (Qiagen v22.0.2; CLC bio, Denmark). To assess the coverage of the raw reads to the MTB reference strain (M. tuberculosis H37Rv, NC_000962.3), the wf-alignment procedure in EPI2ME Labs software (ONT; v0.2.3) and CLC Genomics Workbench were used to evaluate the sequencing depth. Nonsynonymous mutations in the MTB genomic DNA were evaluated and detected by repeatedly using the wf-alignment procedure in EPI2ME Labs software (ONT; v0.2.9) and CLC Genomics Workbench (ONT; v0.2.3).

[0045] 4. Ligand-protein structure modeling and analysis

[0046] The protein structures of pncA (1SJ2), rpsA (4NNI), and panD (6OYY) were downloaded from the protein database (PDBe, 14). The effects of identified variants on the folding and stability of proteins encoded by pncA, rpsA, or panD were simultaneously evaluated using three algorithms: mCSM-Stability, DUET, and SDM. Additionally, the structures of PZA and its active form POA were downloaded from the chemical composition section of PDBe and applied to ligand-protein simulation docking via the Autodock vina website (https: / / vina.scripps.edu / ). Variant calling results generated using CLC Genomics Workbench were fed into a 3D structure modeling pipeline, which simulated the effects of identified variants on drug-target interactions in this invention. The results are shown in Tables 3-5 below:

[0047]

[0048]

[0049] In summary, the molecular markers for drug resistance in Mycobacterium tuberculosis, the reagent kits for detecting drug resistance in Mycobacterium tuberculosis, and the detection methods thereof of the present invention include detecting nonsynonymous 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. This can help doctors select the most suitable treatment plan for pulmonary tuberculosis, improve the success rate of treatment, reduce the infection and spread of pulmonary tuberculosis, thereby protecting patients and public health.

[0050] The embodiments described above are merely for illustrating the technical ideas and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the patent scope of the present invention. All equivalent changes or modifications made in accordance with the spirit disclosed in the present invention should still be covered within the patent scope of the present invention.

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Claims

1. A molecular marker for drug resistance in Mycobacterium tuberculosis, which is the amino acid at position 78 of the pncA protein, which is mutated from glycine to valine: Gly78Val; the drug is pyrazinamide (PZA).

2. The molecular marker as claimed in claim 1, further comprising any one or a combination of the following (E) to (K): (E) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO:9, resulting in the mutated nucleotide sequence shown in SEQ ID NO:10; (F) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO:11, resulting in the mutated nucleotide sequence shown in SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14; (G) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO:15, resulting in the mutated nucleotide sequence 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, resulting in the mutated nucleotide sequence shown in SEQ ID NO:20; (I) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO:21, resulting in the mutated nucleotide sequence shown in SEQ ID NO:22; (J) a mutation occurring based on the nucleotide sequence shown in SEQ ID NO:23, resulting in the mutated nucleotide sequence shown in SEQ ID NO:24; (K) The mutation occurred based on the nucleotide sequence shown in SEQ ID NO:25, and the resulting nucleotide sequence is shown in SEQ ID NO:

26.

3. The molecular marker as described in claim 1, further comprising any one or a combination of the following (E') to (K'): (E') Amino acid at position 175 of the pncA protein, mutated from methionine to lysine: Met175Lys; (F') Amino acid at position 222 of the rpsA protein, mutated from phenylalanine to leucine: Phe222Leu; (G') Amino acid at position 225 of the rpsA protein, mutated from phenylalanine to leucine: Phe225Leu; (H') Amino acid at position 86 of the panD protein, mutated from isoleucine to valine: Ile86Val; (I') Amino acid at position 86 of the panD protein, mutated from isoleucine to sine: Ile86Thr; (J') The 72nd amino acid in the panD protein is mutated from aspartic acid to sine: Asn72Thr; the 90th amino acid in the (K') panD protein is mutated from tyrosine to aspartic acid: Tyr90Asp.

4. A reagent kit for detecting drug resistance in Mycobacterium tuberculosis, used to detect a specific molecular marker, wherein the molecular marker is the amino acid at position 78 of the pncA protein, which is mutated from glycine to valine (Gly78Val); wherein the drug resistance is against pyrazinamide (PZA).

5. A method for detecting drug resistance in Mycobacterium tuberculosis, comprising detecting a specific molecular marker, wherein the molecular marker is an amino acid at position 78 of the pncA protein, which is mutated from glycine to valine (Gly78Val); wherein the drug resistance is against pyrazinamide (PZA).

6. The method for detecting drug resistance in Mycobacterium tuberculosis as described in claim 5, comprising the steps of detecting the gene sequence of Mycobacterium tuberculosis (MTB) in a specimen.

7. The method for detecting drug resistance in Mycobacterium tuberculosis as described in claim 6, wherein the method for detecting the gene sequence of MTB includes gene sequencing technology, which involves comparing the sequencing results with wild-type sequences SEQ ID NO: 9, 11, 15, 19, 21, 23, 25 to confirm the status of the molecular marker.

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