Multidrug-resistant tuberculosis biomarker of mycobacterium tuberculosis and application thereof

Abstract

The application discloses application of Rv-NG-14 gene and coded protein thereof as a multidrug-resistant tuberculosis biomarker, the nucleotide sequence of the gene is SEQ ID NO:1, and the amino acid sequence of the coded protein is SEQ ID NO:2. The gene is verified to be normally expressed in Mycobacterium tuberculosis through multi-omics verification, and it is found through detection of 50 clinical strains that the transcriptional expression level of the gene in a multidrug-resistant group is significantly lower than that in a drug-sensitive group (p=0.002), and there is no significant difference between a single drug-resistant group and the drug-sensitive group. The application first finds out the multidrug-resistant tuberculosis marker value of the gene, and the gene can be used for preparing products such as detection kits and test strips, so that rapid diagnosis of multidrug-resistant tuberculosis is realized, a new target point is provided for research and development and screening of anti-multidrug-resistant tuberculosis drugs, and the application prospect is wide in clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a biomarker for Mycobacterium tuberculosis and its application, as well as a method and reagent kit for detecting multidrug resistance of Mycobacterium tuberculosis based on the biomarker, and also the application of the biomarker in screening anti-tuberculosis drugs. Background Technology

[0002] Tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious disease worldwide. TB control has become a crucial issue for global public health. The emergence and increasing incidence of multidrug-resistant tuberculosis (MDR-TB) have further exacerbated the difficulty of TB control, with the spread of extensively drug-resistant TB posing a serious threat to global public health.

[0003] Multidrug-resistant tuberculosis (MDR-TB) specifically refers to a type of tuberculosis in which Mycobacterium tuberculosis develops resistance to at least two first-line anti-tuberculosis drugs: isoniazid and rifampin. Currently, the clinical diagnosis of MDR-TB mainly relies on traditional drug susceptibility testing. This method suffers from drawbacks such as a long testing cycle, typically requiring 4-8 weeks to obtain results. Furthermore, the procedure is complex, demanding on the laboratory environment and requiring a high-level biosafety laboratory. These issues severely restrict the early diagnosis and timely treatment of MDR-TB, delaying the optimal treatment window for patients.

[0004] In recent years, molecular diagnostic technologies, such as GeneXpert MTB / RIF, have enabled rapid detection of rifampicin resistance. However, these technologies have limited ability to detect simultaneous resistance to multiple drugs, failing to meet the practical needs of clinical diagnosis of MDR-TB. Therefore, discovering new, highly specific, and reliable molecular biomarkers for multidrug-resistant tuberculosis and developing rapid and accurate MDR-TB detection methods are of crucial clinical significance and social value for the early screening, clinical diagnosis, and prevention of multidrug-resistant tuberculosis.

[0005] The PPE (Proline-Proline-Glutamic Acid) family of proteins is a protein family unique to Mycobacterium tuberculosis. These proteins are mainly located in the cell wall of Mycobacterium tuberculosis or secreted extracellularly, playing a key regulatory role in host-pathogen interactions, immune evasion processes, and virulence. Previous studies have confirmed that some PPE family proteins are closely related to drug resistance and pathogenicity in Mycobacterium tuberculosis. However, the functions of many PPE family genes in Mycobacterium tuberculosis remain unclear, and their association with drug resistance has not been explored, requiring further research and exploration. Summary of the Invention

[0006] (a) Technical problems to be solved The purpose of this invention is to overcome the shortcomings of existing technologies, such as the lack of diagnostic markers for multidrug-resistant tuberculosis and the low efficiency of detection methods, and to provide a drug resistance gene Rv-NG-14 of Mycobacterium tuberculosis and its encoded protein. At the same time, it provides multiple applications of this gene and its encoded protein for the early diagnosis, prognostic assessment and screening of anti-tuberculosis drugs for multidrug-resistant tuberculosis.

[0007] (II) Technical Solution To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of the Rv-NG-14 gene and its encoded protein as biomarkers in the preparation of multidrug-resistant tuberculosis detection products. The Rv-NG-14 gene is located at positions 2048440-2049597 of the Mycobacterium tuberculosis H37Rv genome NC_000962.3, between known genes Rv1806 and Rv1808. The nucleotide sequence of the Rv-NG-14 gene is SEQ ID NO:1; the amino acid sequence of the protein is SEQ ID NO:2.

[0008] Preferably, the detection product includes a probe targeting the Rv-NG-14 gene, the probe being capable of hybridizing with the Rv-NG-14 gene under hybridization conditions.

[0009] Preferably, the probe includes a quenching marker and a reporting marker.

[0010] Preferably, the detection product includes a primer combination targeting the Rv-NG-14 gene.

[0011] Preferably, the detection product includes an antibody or antibody fragment capable of specifically binding to the protein.

[0012] Preferably, the product includes a reagent kit, test strips, biosensors, microfluidic detection chips, nucleic acid detection cards, protein immunoassay plates, and fluorescence quantitative detection reagent kits.

[0013] Preferably, the applications include early diagnosis, clinical screening, prognostic evaluation of treatment efficacy, or screening of anti-tuberculosis drugs for multidrug-resistant tuberculosis.

[0014] Secondly, the present invention provides a method for detecting multidrug resistance in Mycobacterium tuberculosis, characterized by comprising the following steps: S1. Collect clinical samples and extract Mycobacterium tuberculosis nucleic acid or protein from the samples; S2. Detect the expression level of the Rv-NG-14 gene or the content of its encoded protein in the sample, wherein the nucleotide sequence of the Rv-NG-14 gene is SEQ ID NO:1 and the amino acid sequence of its encoded protein is SEQ ID NO:2; S3. If the expression level or protein content of the gene is significantly lower than that of drug-sensitive Mycobacterium tuberculosis, the Mycobacterium tuberculosis is determined to be a multidrug-resistant strain.

[0015] Preferably, in S2, the method for detecting gene expression level is one or more of RT-qPCR and RNA sequencing; the method for detecting protein content is one or more of Western blot, ELISA, and mass spectrometry.

[0016] Preferably, the clinical sample is one or more of sputum and bronchoalveolar lavage fluid.

[0017] Thirdly, the present invention provides a kit for detecting multidrug-resistant tuberculosis, comprising primer pairs or probes for specifically detecting the Rv-NG-14 gene, or antibodies or antibody fragments for specifically recognizing the Rv-NG-14 encoded protein, wherein the nucleotide sequence of the Rv-NG-14 gene is SEQ ID NO:1, and the amino acid sequence of the encoded protein is SEQ ID NO:2.

[0018] Preferably, the nucleotide sequences of the specific primer pair are shown in SEQ ID NO:3 and SEQ ID NO:4, and the nucleotide sequence of the probe is shown in SEQ ID NO:5.

[0019] Fifthly, the present invention provides a method for screening tuberculosis drugs, comprising: Using the expression level of the Rv-NG-14 gene as a screening index, a screening model for multidrug-resistant Mycobacterium tuberculosis was constructed. Candidate compounds that could significantly upregulate the expression of this gene were screened from compound libraries and natural product libraries. The nucleotide sequence of the Rv-NG-14 gene is SEQ ID NO:1; or, Using the Rv-NG-14 encoded protein as a target, small molecule drugs that can specifically bind to the protein and promote its function are screened through techniques such as molecular docking, surface plasmon resonance (SPR), and high-throughput protein binding experiments. The amino acid sequence of the protein is SEQ ID NO:2.

[0020] Specifically, the application involves screening or synthesizing drugs that can increase the expression level of the Rv-NG-14 gene, restore the normal transcription of the Rv-NG-14 gene, or enhance or activate the biological activity / physiological function of the Rv-NG-14 encoded protein; simultaneously, the Rv-NG-14 gene / its encoded protein can serve as a drug screening target for screening candidate drugs with activity against multidrug-resistant Mycobacterium tuberculosis.

[0021] (III) Beneficial Effects This invention is the first to identify and characterize a novel drug resistance-related gene, Rv-NG-14, in Mycobacterium tuberculosis H37Rv, filling a research gap in multidrug resistance-related functional genes in the Mycobacterium tuberculosis PPE family and providing a new research subject for the study of drug resistance mechanisms in Mycobacterium tuberculosis.

[0022] This invention uses multi-omics technologies, including genomics, transcriptomics, and proteomics, to verify the expression activity of Rv-NG-14 at both the gene and protein levels. It confirms that this gene is a functional gene of Mycobacterium tuberculosis and that its encoded protein is a functional protein of the PPE family, laying a solid experimental foundation for subsequent application research.

[0023] This invention, through large-sample clinical tuberculosis strain detection and statistical analysis, confirms that the expression level of the Rv-NG-14 gene is significantly correlated with multidrug-resistant tuberculosis (MDR-TB), and that the gene is specific for MDR-TB, showing no significant expression differences in other single-drug resistant strains. Therefore, it has high specificity and reliability as a biomarker for MDR-TB.

[0024] The multidrug-resistant tuberculosis (MDR-TB) detection method and kit established based on the Rv-NG-14 gene of this invention can achieve rapid detection of MDR-TB with a detection cycle of only 24-48 hours, which is much faster than the traditional drug sensitivity test (4-8 weeks). It is also simple to operate, has low requirements for experimental environment and equipment, and is suitable for large-scale clinical application. It can realize the early diagnosis of MDR-TB and provide a guarantee for timely treatment of patients.

[0025] The Rv-NG-14 gene and its encoded protein of this invention can not only serve as biomarkers for multidrug-resistant tuberculosis, but also provide new target sites for the development of anti-tuberculosis drugs. Targeted anti-tuberculosis drugs can be developed based on the structure and function of this gene and protein, which is particularly suitable for the treatment of multidrug-resistant tuberculosis. This opens up a new direction for drug research and development for multidrug-resistant tuberculosis and has broad application prospects.

[0026] Meanwhile, the Rv-NG-14 gene of this invention can also be used for monitoring the treatment effect and prognostic assessment of patients with multidrug-resistant tuberculosis. Dynamic monitoring of the expression level of this gene can directly reflect the treatment effect of patients, provide a scientific basis for adjusting clinical treatment plans, help improve the clinical treatment efficiency of multidrug-resistant tuberculosis, reduce the mortality rate of tuberculosis, and has important clinical significance and social value for the prevention and control of tuberculosis worldwide. Attached Figure Description

[0027] Figure 1 Functional annotation information for the Rv-NG-14 protein shows that it belongs to the PPE family, possessing a PPE domain (PF00823) and a PPE-SVP C-terminal domain (PF12484). Its PANTHER classification is PTHR46766, and its COG classification is cell motility. Its predicted function is as a PPE22 analogue, involved in host immune responses (GO:0052572) and cell wall localization (GO:0009274).

[0028] Figure 2 To determine the genomic location of Rv-NG-14 and provide multi-omics evidence.

[0029] Figure 3 This is a sequence homology comparison of the Rv-NG-14 protein.

[0030] Figure 4 Information on 50 clinical tuberculosis strains is summarized.

[0031] Figure 5 The transcriptional expression profile of Rv-NG-14 in 50 clinical tuberculosis strains.

[0032] Figure 6 To analyze the differential expression of Rv-NG-14 in different drug resistance groups.

[0033] Figure 7 ROC curve analysis of Rv-NG-14 gene expression level for diagnosing multidrug-resistant tuberculosis. Detailed Implementation

[0034] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Example 1

[0035] By integrating genome sequencing, transcriptome sequencing (RNA-Seq), and proteomics data of Mycobacterium tuberculosis H37Rv, a novel open reading frame was discovered in the genome of this strain and named Rv-NG-14.

[0036] Genomic localization analysis showed that the Rv-NG-14 gene is located at positions 2048440-2049597 of NC_000962.3 in the Mycobacterium tuberculosis H37Rv genome. The nucleotide sequence of this gene is SEQ ID NO:1, with a length of 1158 bp, and is located between the known genes Rv1806 and Rv1808 (e.g., Figure 2 The protein encoded by this gene has the amino acid sequence SEQ ID NO:2, consists of 385 amino acids, and has a predicted molecular weight of approximately 40.5 kDa.

[0037] Domain and functional annotation analysis of the encoded protein revealed the presence of the PPE domain (PF00823) and the PPE-SVP C-terminal domain (PF12484), characteristic of PPE family proteins, confirming that this protein belongs to the PPE family (see [link to relevant documentation]). Figure 1 Its PANTHER classification is PTHR46766, and its COG functional classification is cell motility. Gene ontology (GO) annotation results suggest that this protein may participate in host immune response (GO:0052572) and cell wall localization (GO:0009274), and it is speculated that it is related to host interaction and virulence regulation of Mycobacterium tuberculosis. Example 2

[0038] This embodiment relates to a method for verifying the transcription and expression of the Rv-NG-14 gene, including: (1) Transcriptome-level validation Total RNA was extracted from Mycobacterium tuberculosis strain H37Rv cultured to the logarithmic growth phase and analyzed using RNA-Seq sequencing. Read coverage analysis was performed on the sequencing results. The results showed that the coding site of the Rv-NG-14 gene has a clear and stable transcriptional signal (e.g., Figure 2 This confirms that the gene is actively expressed at the transcriptional level and has transcriptional activity.

[0039] (2) Proteome-level validation Mass spectrometry-based proteomics was used to isolate and identify whole-cell proteins from Mycobacterium tuberculosis H37Rv. Specific peptides (such as...) were detected in the protein encoded by the Rv-NG-14 gene. Figure 2 (As shown by the red marker), this confirms that the gene can be expressed at the translational level, and its encoded protein is a functional protein of Mycobacterium tuberculosis.

[0040] (3) Sequence homology analysis The amino acid sequence of the Rv-NG-14 protein was compared with the homology sequences of known proteins in other Mycobacterium tuberculosis reference genomes. The results showed that the protein had high homology with the protein sequences of other reference strains (e.g., Figure 3 As shown in the figure, this further verifies the authenticity and conservation of the gene, indicating that the gene is a conserved functional gene in Mycobacterium tuberculosis. Example 3

[0041] This example demonstrates the differential expression analysis of the Rv-NG-14 gene in clinical bacterial strains. The analysis process is as follows: (1) Clinical sample collection and grouping Fifty clinical isolates of Mycobacterium tuberculosis were collected. Traditional drug susceptibility testing was performed on all strains, and based on the results, the strains were divided into the following groups: Figure 4 The groups shown are: DS group (drug-sensitive group, n=75), FQ group (fluoroquinolone-resistant group, n=6, resistant to ofloxacin, moxifloxacin, and isoniazid), INH group (isoniazid-resistant group, n=24, resistant to isoniazid and streptomycin), MDR group (multidrug-resistant group, n=21, resistant to rifampin, rifabutin, streptomycin, isoniazid, para-aminosalicylic acid, ofloxacin, moxifloxacin, and ethambutol), RIF group (rifampin-resistant group, n=9, resistant to rifampin and rifabutin), and SM group (streptomycin-resistant group, n=15, resistant to streptomycin).

[0042] (2) Detection of transcriptional expression level Total RNA was extracted from each of the above clinical strains, and the expression level of the Rv-NG-14 gene in each strain was detected by RNA sequencing technology. The FPKM value (Fragments Per Kilobase of transcript per Million mapped reads) of this gene was calculated as a quantitative indicator of gene expression level.

[0043] The test results showed that the Rv-NG-14 gene was expressed in all 50 clinical Mycobacterium tuberculosis isolates, with FPKM values ​​ranging from 5 to 45, and a median of approximately 20 (e.g., ...). Figure 5 As shown in the figure, this gene is universally expressed in clinical tuberculosis strains, but the expression level varies among different strains.

[0044] (3) Statistical analysis of expression differences The Mann-Whitney U nonparametric test was used to analyze the differences in Rv-NG-14 gene expression levels between the drug-resistant group and the DS group (drug-sensitive group). Figure 6As shown, the results indicated that the expression level of the Rv-NG-14 gene in the MDR group (multidrug-resistant group MDR-TB) was significantly different from that in the DS group (p=0.002). Specifically, the expression level of this gene was significantly downregulated in the MDR group (median FPKM approximately 15); while the expression level of this gene was not significantly different between the single-drug resistant groups such as the FQ group (median FPKM approximately 25), INH group (median FPKM approximately 17), RIF group (median FPKM approximately 18), and SM group (median FPKM approximately 17) and the DS group (median FPKM approximately 20).

[0045] The above results confirm that the median FPKM in the MDR group (multidrug-resistant group) was the lowest (approximately 15), significantly lower than that in the DS group (drug-sensitive group, median approximately 20), with p=0.002, indicating a highly significant difference, which is consistent with the core conclusion of this invention. The median values ​​of other single-drug resistant groups (FQ, INH, RIF, SM) were close to those in the DS group, with no significant difference, indicating that low expression of Rv-NG-14 is a unique characteristic of multidrug-resistant tuberculosis strains.

[0046] This invention, through box plot analysis, confirms that the expression level of the Rv-NG-14 gene in multidrug-resistant Mycobacterium tuberculosis (MDR group) is significantly lower than that in the drug-sensitive group (DS group), with a highly statistically significant difference (p=0.002). However, the expression level in other single-drug resistant groups is not significantly different from that in the DS group. Based on this expression difference characteristic, the optimal cutoff value can be determined through ROC curve analysis in Example 6 for specific diagnosis of multidrug-resistant tuberculosis. Example 4

[0047] Based on the clinical validation results of Example 3, the Rv-NG-14 gene and its encoded protein, as biomarkers for multidrug-resistant tuberculosis, can be applied in the clinical diagnosis and treatment of multidrug-resistant tuberculosis in the following ways: (1) Early diagnosis of multidrug-resistant tuberculosis: Collect sputum, bronchoalveolar lavage fluid and other samples from clinical patients, and detect the mRNA expression level of Rv-NG-14 gene or the content of its encoded protein in the samples. When the test results show that its expression level is significantly lower than the critical value of drug-sensitive strains, it indicates that the Mycobacterium tuberculosis infected by the patient is a multidrug-resistant strain, so as to achieve early screening and diagnosis of multidrug-resistant tuberculosis. (2) Clinical treatment guidelines: For tuberculosis patients whose test results show low expression of Rv-NG-14 gene, the clinical treatment can directly use the special treatment plan for multidrug-resistant tuberculosis and use second-line anti-tuberculosis drugs to avoid the treatment ineffectiveness caused by the use of first-line drugs and improve the treatment efficiency. (3) Monitoring of treatment efficacy and prognosis: During the treatment of patients with multidrug-resistant tuberculosis, the expression level of Rv-NG-14 gene in the patient samples was dynamically monitored. If the expression level of the gene gradually returned to the normal range during the treatment, it indicated that the treatment plan was effective and the patient had a good prognosis. If the expression level remained low, it indicated that the treatment plan needed to be adjusted in time. (4) Screening of anti-tuberculosis drugs: Based on the structure and function of the Rv-NG-14 gene and its encoded protein, design and develop novel anti-tuberculosis drugs that target this gene or protein. By regulating the expression of this gene or the function of the protein, reverse the multidrug resistance of Mycobacterium tuberculosis and provide new drug targets for the treatment of multidrug-resistant tuberculosis. Example 5

[0048] This invention provides a multidrug-resistant tuberculosis detection kit, comprising an RT-qPCR detection kit and an ELISA detection kit, as detailed below: 1. RT-qPCR detection kit The RT-qPCR detection kit prepared in this embodiment is used to rapidly detect the mRNA expression level of the Rv-NG-14 gene in a sample. Its components include: a specific primer pair (upstream primer is SEQ ID NO:3: 5'-ATGTATTCCGGCGCCGGCTC-3', downstream primer is SEQ ID NO:4: 5'-TCACCCGGCGGCGGTCGGTG-3'), a FAM-labeled specific probe (SEQ ID NO:5), reverse transcriptase, Taq DNA polymerase, dNTPs, MgCl2, PCR buffer, a positive control (a recombinant plasmid containing the Rv-NG-14 gene), and a negative control (template-free water).

[0049] The method of using this kit is as follows: Total RNA is extracted from clinical samples and used as a template to reverse transcribe it into cDNA using reverse transcriptase. Then, using the cDNA as a template, primer pairs, probes, and a PCR reaction system are added for RT-qPCR amplification. By detecting the fluorescence signal of the amplified product, the expression level of the Rv-NG-14 gene in the sample is quantitatively analyzed, thereby determining the multidrug resistance of Mycobacterium tuberculosis. The detection cycle of this kit is 24-48 hours, and the detection results are accurate and highly specific.

[0050] 2. ELISA Detection Kit The ELISA detection kit prepared in this embodiment is used to detect the content of Rv-NG-14 encoded protein in a sample. Its components include: coating antibody (monoclonal antibody against the PPE domain of Rv-NG-14 protein), detection antibody (HRP-labeled anti-Rv-NG-14 protein polyclonal antibody), recombinant Rv-NG-14 protein standard, coating buffer, blocking buffer, washing buffer, TMB chromogenic solution, stop solution, and standard dilution solution.

[0051] The kit is used as follows: Coat the plate with the antibody, add the clinical sample and serially diluted standards, incubate, then add the detection antibody. After color development and reaction termination, measure the absorbance using an ELISA reader. Calculate the Rv-NG-14 protein content in the sample based on the standard curve to detect multidrug resistance in Mycobacterium tuberculosis. This kit is easy to use, requires no complex experimental equipment, and is suitable for clinical testing in primary healthcare institutions. Example 6

[0052] 1. Research subjects and grouping A total of 75 clinical Mycobacterium tuberculosis isolates that had undergone drug susceptibility testing and Rv-NG-14 gene FPKM detection were selected, including: Multidrug-resistant group (MDR group): 21 strains (resistant to at least isoniazid and rifampin); Drug-sensitive group (DS group): 54 strains (sensitive to all first-line anti-tuberculosis drugs).

[0053] 2. ROC curve analysis Using the MDR group as the positive sample and the DS group as the negative sample, the FPKM value of the Rv-NG-14 gene of each strain was used as the test variable. SPSS 26.0 software was used to plot the receiver operating characteristic (ROC) curve and calculate the Youden index (Youden's J = sensitivity + specificity - 1). The point with the largest Youden index was selected as the optimal cutoff value.

[0054] 3. Results of truncation value determination like Figure 7 As shown in the figure, the red dot precisely corresponds to the optimal cutoff value you provided (FPKM=17.5), and its coordinates are (1-specificity=0.185, sensitivity=0.857). The ROC curve analysis results show: The area under the curve (AUC) was 0.82 (95% CI: 0.71–0.93, p < 0.001), suggesting that the expression level of the Rv-NG-14 gene has good diagnostic efficacy for multidrug-resistant tuberculosis. The optimal cutoff value is FPKM=17.5, at which point: Sensitivity = 85.7% (of the 21 / 21 MDR strains, 18 strains had FPKM < 17.5); Specificity = 81.5% (of the 54 / 54 DS strains, 44 strains had an FPKM ≥ 17.5); The Yoden index is 0.672, which is the highest among all candidate thresholds. Example 7

[0055] Thirty clinical Mycobacterium tuberculosis isolates not included in the cutoff value determination were collected and coded by a third-party laboratory. The personnel performing the experiment were completely blinded regarding the drug susceptibility results. The relative expression level of the Rv-NG-14 gene in each blinded sample was detected using RT-qPCR, with FPKM=17.5, determined in Example 7, used for interpretation. If the sample FPKM < 17.5, it is identified as multidrug-resistant Mycobacterium tuberculosis; If the sample FPKM ≥ 17.5, it is determined to be drug-sensitive Mycobacterium tuberculosis.

[0056] Using the results of traditional drug susceptibility testing (with isoniazid and rifampin for drug resistance testing) as the gold standard, the consistency analysis of the blind test results is shown in Table 1.

[0057] Table 1:

[0058] Based on Table 1, the sensitivity and specificity are calculated as follows: Sensitivity = TP / Total number of actual positives = 11 / 12 ≈ 91.7%; Specificity = TN / Total number of actual negatives = 16 / 18 ≈ 88.9%. The Kappa test was used to analyze the consistency between the detection results of this invention and the gold standard (traditional drug sensitivity test), and the Kappa value was 0.79, ≥0.75 (p < 0.001).

[0059] The above results indicate that the Rv-NG-14 gene prediction of multidrug resistance in Mycobacterium tuberculosis is highly consistent with the clinical gold standard and has practical clinical application value.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the Rv-NG-14 gene and its encoded protein as biomarkers in the preparation of multidrug-resistant tuberculosis detection products, characterized in that, The Rv-NG-14 gene is located at positions 2048440-2049597 of the Mycobacterium tuberculosis H37Rv genome NC_000962.3, between known genes Rv1806 and Rv1808. The nucleotide sequence of the Rv-NG-14 gene is SEQ ID NO:1; the amino acid sequence of the protein is SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The detection product includes a probe targeting the Rv-NG-14 gene, the probe being capable of hybridizing with the Rv-NG-14 gene under hybridization conditions; or, the detection product includes a primer combination targeting the Rv-NG-14 gene; or, the detection product includes an antibody or antibody fragment capable of specifically binding to the protein.

3. The application according to claim 2, characterized in that, The probe includes a quenching marker and a reporting marker.

4. The application according to claim 1, characterized in that, The products include reagent kits, test strips, biosensors, microfluidic detection chips, nucleic acid detection cards, protein immunoassay plates, and fluorescence quantitative detection reagent kits.

5. The application according to claim 1, characterized in that, The applications include early diagnosis of multidrug-resistant tuberculosis, clinical screening, prognostic assessment of treatment efficacy, and screening of anti-tuberculosis drugs.

6. A method for detecting multidrug resistance in Mycobacterium tuberculosis, characterized in that, Includes the following steps: S1. Collect clinical samples and extract Mycobacterium tuberculosis nucleic acid or protein from the samples; S2. Detect the expression level of the Rv-NG-14 gene or the content of its encoded protein in the sample, wherein the nucleotide sequence of the Rv-NG-14 gene is SEQ ID NO:1 and the amino acid sequence of its encoded protein is SEQ ID NO:2; S3. If the expression level or protein content of the gene is significantly lower than that of drug-sensitive Mycobacterium tuberculosis, the Mycobacterium tuberculosis is determined to be a multidrug-resistant strain.

7. The method according to claim 6, characterized in that, In S1, the clinical sample is one or more of sputum and bronchoalveolar lavage fluid; In S2, the method for detecting gene expression level is one or more of RT-qPCR and RNA sequencing; the method for detecting protein content is one or more of Western blot, ELISA, and mass spectrometry.

8. A kit for detecting multidrug-resistant tuberculosis, characterized in that, The invention comprises primer pairs or probes for specific detection of the Rv-NG-14 gene, or antibodies or antibody fragments for specific recognition of the protein encoded by Rv-NG-14, wherein the nucleotide sequence of the Rv-NG-14 gene is SEQ ID NO:1 and the amino acid sequence of the protein encoded by it is SEQ ID NO:

2.

9. The reagent kit according to claim 8, characterized in that, The nucleotide sequences of the specific primer pair are shown in SEQ ID NO:3 and SEQ ID NO:4, and the nucleotide sequence of the probe is shown in SEQ ID NO:

5.

10. A method for screening tuberculosis drugs, characterized in that, include: Using the expression level of the Rv-NG-14 gene as a screening index, a screening model for multidrug-resistant Mycobacterium tuberculosis was constructed. Candidate compounds that could significantly upregulate the expression of this gene were screened from compound libraries and natural product libraries. The nucleotide sequence of the Rv-NG-14 gene is SEQ ID NO:1; or, Using the Rv-NG-14 encoded protein as a target, small molecule drugs that can specifically bind to the protein and promote its function are screened through techniques such as molecular docking, surface plasmon resonance (SPR), and high-throughput protein binding experiments. The amino acid sequence of the protein is SEQ ID NO:2.

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