Drug resistance marker based on 7-methylguanosine modification level of mycobacterium tuberculosis rna and application thereof

Abstract

This invention relates to molecular diagnostic techniques for tuberculosis, specifically to a drug resistance marker based on the level of 7-methylguanosine modification on Mycobacterium tuberculosis RNA and its application. The marker is 7-methylguanosine m-modified guanosine on Mycobacterium tuberculosis RNA. 7 G-modification, m in drug-resistant Mycobacterium tuberculosis RNA 7 The level of G modification is positively correlated with the drug resistance of Mycobacterium tuberculosis. The drug resistance biomarker of this invention enables rapid, accurate, and early drug resistance detection, and the biomarker's drug resistance prediction has the characteristics of wider drug coverage and better versatility, meeting the clinical need for rapid, broad-spectrum, and efficient screening for multidrug resistance in Mycobacterium tuberculosis.

Description

Technical Field

[0001] This invention relates to molecular diagnostic techniques for tuberculosis, specifically to a drug resistance marker based on the level of 7-methylguanosine modification of Mycobacterium tuberculosis RNA and its application. Background Technology

[0002] Mycobacterium tuberculosis is the pathogen that causes tuberculosis. Drug-resistant tuberculosis, especially multidrug-resistant tuberculosis (MDR-TB), has become a major challenge for global tuberculosis control. At present, clinical drug resistance detection mainly relies on phenotypic drug susceptibility testing and gene mutation detection, which have the following drawbacks: (1) the detection cycle is long, requiring 7-28 days; (2) it can only detect a limited number of gene loci and cannot cover all drug resistance mechanisms; (3) the operation is complicated and costly, making it difficult to meet the needs of rapid diagnosis.

[0003] Furthermore, current methods for detecting drug resistance genes in Mycobacterium tuberculosis mostly target only a single drug type, lacking universal biomarkers that can broadly predict resistance to most antimicrobial drugs, thus limiting their applicability and clinical practicality. Existing technologies, such as patent CN109234414B, only target resistance to para-aminosalicylic acid, obtaining Rv3890c, Rv2002, Rv1886c, Rv3824c, and Rv3825c as diagnostic biomarkers through genomic methylation and expression profiling, only addressing the issue of time-consuming single-drug resistance detection; patent CN108950031A only detects rifampicin resistance by measuring the expression levels of three genes, Rv0840c, Rv2386c, and Rv1140, still exhibiting the drawbacks of single-target targeting and limited drug coverage; patent CN118792431B only focuses on gyrA and gyrB mutation sites related to fluoroquinolones, failing to cover common multidrug resistance scenarios in clinical practice. In summary, existing drug resistance detection technologies generally suffer from problems such as narrow drug coverage, single biomarker, and poor universality, making it difficult to meet the clinical needs for rapid, broad-spectrum, and efficient screening for multidrug resistance in Mycobacterium tuberculosis.

[0004] RNA methylation modification is an important post-transcriptional regulatory mechanism in pathogens, playing a key role in their growth, metabolism, and drug resistance. 7-Methylguanosine (m...) 7 G) modification is one of the most common methylation modifications on RNA, but there is currently no way to modify m 7 Reports on the use of G modification for drug resistance detection in Mycobacterium tuberculosis. Summary of the Invention

[0005] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method based on 7-methylguanosine (m 7G) Modified biomarkers, methods, and kits for detecting drug resistance in Mycobacterium tuberculosis enable rapid, accurate, and early detection of drug resistance. The biomarkers also offer advantages such as broader drug coverage and greater versatility in predicting drug resistance, meeting the clinical need for rapid, broad-spectrum, and efficient screening for multidrug resistance in Mycobacterium tuberculosis.

[0006] (II) Technical Solution The technical solution of the present invention is as follows: In a first aspect, the present invention provides a biomarker for detecting drug resistance in Mycobacterium tuberculosis, wherein the biomarker is 7-methylguanosine (m...) on Mycobacterium tuberculosis RNA. 7 G) Modification, m in drug-resistant Mycobacterium tuberculosis RNA 7 The level of G modification is positively correlated with the drug resistance of Mycobacterium tuberculosis.

[0007] Preferably, m from the RNA of drug-sensitive strains 7 The G modification level is used as a control baseline: when the RNA of the drug-resistant strain contains m 7 When the G modification level is ≥ 1.4 times (140%) of the control baseline, it is determined to be: drug-resistant Mycobacterium tuberculosis.

[0008] The drug resistance types of the Mycobacterium tuberculosis include: (1) Resistance to first-line anti-tuberculosis drugs, including at least one of isoniazid, rifampin, ethambutol and pyrazinamide; (2) Resistance to second-line anti-tuberculosis drugs, including at least one of fluoroquinolones, bedaquiline, linezolid, clofazimine, streptomycin, kanamycin and amikacin; The drug resistance types include single drug resistance, multiple drug resistance, multidrug resistance (MDR-TB), and extensively drug-resistant (XDR-TB).

[0009] Secondly, the present invention also provides a method for detecting drug resistance in Mycobacterium tuberculosis, comprising the following steps: (1) Extract total RNA from the test strain; (2) Detect the modification level of 7-methylguanosine in RNA; (3) Compare the detection results with the m in the RNA of drug-sensitive Mycobacterium tuberculosis. 7 G-level comparison; (4) According to m 7 An elevated G modification level indicates a drug-resistant strain.

[0010] Preferably, the detection method employs liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0011] Preferably, in step (4), the m from the RNA of the drug-sensitive strain is used. 7The G modification level is used as a control baseline: when the RNA of the drug-resistant strain contains m 7 When the G modification level is ≥ 1.4 times (140%) of the control baseline, it is determined to be: drug-resistant Mycobacterium tuberculosis.

[0012] Preferably, the detection method includes: Total RNA from the test strain was extracted using the Trizol method or an RNA extraction kit to remove residual genomic DNA; the extracted RNA was digested with a nucleic acid digestion mixture to convert the RNA into mononucleotides in a one-step process; commercially available RNA m 7 Using G (Cat.NO: 20244-86-4) and rG (Cat.NO: 118-00-3) standards as references, standard solutions were prepared, and a standard detection method was established based on the multiple reaction monitoring (MRM) technology of Xevo TQ triple quadrupole mass spectrometry. Among them: rG collected 284.1 m / z precursor ions, 152.1 m / z fragment ions, and retained them for 3.55 min; m 7 G collected precursor ions at 298.1 m / z, fragment ions at 166.1 m / z, with a retention time of 2.46 min; collision energy at 16 V. Based on the established standard MRM procedure, targeted mass spectrometry data acquisition was performed on the mononucleotides obtained from the enzyme digestion of the test strains. By comparing the concentrations of standards, the m-values ​​of RNA molecules in the strains were accurately determined. 7 The G / rG ratio, which is m 7 The relative abundance of G, where a higher relative abundance indicates higher m. 7 The higher the level of G modification.

[0013] Furthermore, using the standard strain of Mycobacterium tuberculosis as a control, the concentration was 1.4 times higher than the control, and was thus determined to be m. 7 A high level of G modification indicates high drug resistance in this strain; if the level is lower than or equal to the control, it is considered m. 7 The strain with a low level of G modification corresponds to low drug resistance.

[0014] Secondly, the present invention also provides a method based on m 7 G-modified assay kit for detecting drug resistance in Mycobacterium tuberculosis, containing a reagent for detecting 7-methylguanosine (m 7 The test reagent (G) is used for the detection of drug resistance in Mycobacterium tuberculosis.

[0015] (III) Beneficial Effects This invention demonstrated in 50 clinical strains of Mycobacterium tuberculosis that the m of drug-resistant strains 7 The G modification level was significantly higher in the G strain than in the susceptible strain, while m 3The modification levels of G did not differ significantly. Therefore, m 7 G-modification can serve as a molecular marker for drug resistance in Mycobacterium tuberculosis, providing a novel direction for drug resistance detection and new drug targets. This invention uses 50 clinical strains (25 resistant + 25 susceptible), including multidrug-resistant and multidrug-resistant (MDR-TB) strains, covering almost all conventional tuberculosis drugs. Therefore, compared to existing biomarkers, it has a wider coverage of drugs for predicting drug resistance and better versatility, meeting the clinical need for rapid, broad-spectrum, and efficient screening for multidrug resistance in Mycobacterium tuberculosis, and can be used to guide precision medicine for tuberculosis. Attached Figure Description

[0016] Figure 1 The experiment in Example 1 revealed m of 25 drug-resistant strains. 7 The relative abundance of G was significantly higher than that of the 25 drug-sensitive strains (p=0.046).

[0017] Figure 2 The experiment in Example 2 revealed a difference in m between the SM group and the sensitive group (DS). 7 There was a significant difference in G abundance (p=0.0225) between the FQ group and the sensitive group (DS), with m 7 There were significant differences in G abundance (p=0.0228). Detailed Implementation

[0018] 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.

[0019] This invention is the first to discover and confirm that 7-methylguanosine (m) on Mycobacterium tuberculosis RNA 7 G) The level of modification is closely related to the drug resistance of bacterial strains and can serve as a novel molecular marker for assessing drug resistance in Mycobacterium tuberculosis. Unlike traditional gene mutation detection, which targets only a single drug, m 7 The G modification level can broadly reflect the overall drug resistance status of the strain, enabling unified prediction and typing of resistance to common first-line and second-line anti-tuberculosis drugs in clinical practice. This overcomes the shortcomings of existing technologies, which can only detect specific drugs, have narrow coverage, and poor versatility.

[0020] This invention provides a biomarker for detecting drug resistance in Mycobacterium tuberculosis, wherein the biomarker is 7-methylguanosine (m) in total RNA of Mycobacterium tuberculosis. 7 G) Modification level or relative abundance.

[0021] The m 7 G modification refers to the methylation modification that occurs at the 7th N atom of the guanosine (G) base in RNA after Mycobacterium tuberculosis transcribes RNA using its own DNA as a template. It belongs to post-transcriptional regulatory modification.

[0022] This invention utilizes a systematic testing of 50 clinical Mycobacterium tuberculosis isolates (25 of which were drug-resistant and 25 were drug-sensitive) to confirm that: 1) m in the RNA of drug-resistant strains 7 The G modification level was significantly higher than that of drug-sensitive strains, and the difference was statistically significant (p=0.046). 2) m 7 The level of G modification was positively correlated with the drug-resistant phenotype of Mycobacterium tuberculosis; 3) m 7 The level of G modification can serve as an effective molecular indicator for distinguishing between drug-resistant and drug-sensitive strains.

[0023] When predicting whether Mycobacterium tuberculosis is a durable or non-drug-resistant strain, the test samples used include, but are not limited to: clinical isolates of Mycobacterium tuberculosis, patient sputum samples, Mycobacterium tuberculosis cultures, and total RNA extracted from the samples.

[0024] This invention provides two detection methods that can be used for patent protection: Method 1: Quantitative detection of m by LC-MS / MS (MRM mode) 7 The relative abundance of G-nucleotides is determined through the following steps: 1) Extract total RNA from the sample; 2) Enzymatically break down RNA into mononucleotides; 3) Detection was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) in multiple reaction monitoring (MRM) mode; 4) with m 7 Quantification of G nucleoside standard, calculation of m 7 G relative abundance (m 7 G / rG); 5) Compare with drug-sensitive strains to determine drug resistance.

[0025] Method 2: AlkAniline-Seq sequencing to detect m 7 G site and modification level, as determined by the following method: 1) Extract total RNA and remove rRNA; 2) AlkAniline sequencing was used to sequence m 7 Single-base resolution detection is performed at the G site; 3) Calculate the termination rate, normalized cutoff score, and enrichment factor to obtain m. 7 G modification level; 4) Determine the antibiotic resistance of the strain based on the level of modification.

[0026] The biomarkers provided by this invention can be applied to: 1) early prediction, rapid screening, and auxiliary diagnosis of Mycobacterium tuberculosis drug resistance; 2) Mycobacterium tuberculosis drug resistance typing (single-drug resistance, multi-drug resistance, multidrug resistance, extensively drug-resistant); 3) clinical efficacy monitoring of anti-tuberculosis drugs; 4) screening of novel anti-tuberculosis drug targets (targeting m 7 5) Precision medication guidance for tuberculosis.

[0027] This invention discloses 7-methylguanosine (m) from Mycobacterium tuberculosis. 7 The application of G)RNA modification in drug resistance prediction. This invention is the first to demonstrate the application of G)RNA modification in drug resistance prediction of drug-resistant Mycobacterium tuberculosis. 7 The G modification level was significantly higher than that of drug-sensitive strains, and m 7 The level of G modification was positively correlated with the drug resistance phenotype of the strain. This was achieved by detecting m in Mycobacterium tuberculosis RNA. 7 The relative abundance of G modification can quickly, accurately, and broadly determine whether the tested strain is a drug-resistant or drug-sensitive strain, enabling the prediction, typing, and auxiliary diagnosis of drug resistance in Mycobacterium tuberculosis. Example 1

[0028] By comparing m in drug-resistant and drug-sensitive strains 7 The difference in the level of G modification yields m 7 G modification levels can serve as molecular markers for predicting drug resistance in Mycobacterium tuberculosis.

[0029] (1) Strains and sample information Fifty clinical isolates of Mycobacterium tuberculosis (Mtb) were collected from the clinical laboratory departments of local designated tuberculosis treatment hospitals. These isolates were sputum samples from inpatients and outpatients collected between January 2021 and December 2024. Among them, 25 were drug-resistant strains and 25 were drug-sensitive strains.

[0030] Inclusion criteria: positive for Mycobacterium tuberculosis by Roche culture and nucleic acid amplification (PCR); drug resistance typing of strains completed by absolute concentration method drug susceptibility testing; Grouping: Drug-resistant strains group: 25 strains (including single-drug resistant, multi-drug resistant, and multi-drug resistant strains) Drug-sensitive strains group: 25 strains All strains were stored in glycerol cryovials at -80℃.

[0031] (2) Main instruments and reagents Instruments: Clean bench, constant temperature and humidity incubator (37℃, 5% CO2), high-speed refrigerated centrifuge (4℃, 12000r / min), NanoDrop micro-volume nucleic acid analyzer, LC-MS / MS liquid chromatography-tandem mass spectrometry (AB SCIEX). Reagents: TRIzol RNA extraction kit, nuclease P1, alkaline phosphatase (for RNA digestion), m 7 G nucleoside standard (≥99%, Catalog No.: 20244-86-4), methanol, acetonitrile (chromatographic grade), and G (guanosine) standard (Catalog No.: 118-00-3).

[0032] (3) Experimental steps Step 1: Strain resuscitation and culture Take the strain frozen at -80℃ and inoculate it onto the slant of Löwenstein medium; place it in an incubator at 37℃, 5% CO2, and 60% relative humidity and incubate in the dark for 4 weeks; when typical pale yellow colonies grow on the slant, verify the purity of the colony.

[0033] Step 2: Bacterial cell collection and washing Rinse the culture slant with 5 mL of sterile physiological saline to elute the bacterial cells; transfer the bacterial solution to a sterile centrifuge tube and centrifuge at 12000 r / min, 4℃ for 10 min, and discard the supernatant; add 5 mL of pre-cooled sterile PBS to resuspend and wash, centrifuge at 12000 r / min, 4℃ for 10 min, and repeat the washing twice; finally collect the bacterial pellet and pre-freeze at -80℃ for later use.

[0034] Step 3: Total RNA extraction Add 1 mL of TRIzol lysis buffer to the bacterial cell pellet, vortex thoroughly for 1 min, and incubate at room temperature for 5 min to lyse. Add 200 μL of chloroform, vortex vigorously for 15 s, and incubate at room temperature for 2 min. Centrifuge at 12000 rpm and 4 °C for 15 min, and transfer 400 μL of the upper aqueous phase to a new RNase-free centrifuge tube. Add an equal volume of isopropanol, gently invert to mix, and incubate at room temperature for 10 min. Centrifuge at 12000 rpm and 4 °C for 10 min, discard the supernatant, and wash the RNA pellet with 1 mL of 75% ethanol. After washing, centrifuge at 7500 rpm and 4 °C for 5 min, discard the ethanol, and air dry at room temperature for 5 min. Add 30 μL of RNase-free water to dissolve the RNA, and heat in a 60 °C water bath for 10 min to promote dissolution.

[0035] Step 4: RNA purity and concentration detection Take 1 μL of RNA and determine its concentration and purity using a Nano Drop assay. Acceptable standards: A260 / A280 = 1.8-2.1, A260 / A230 ≥ 2.0 Qualified RNA should be stored at -80°C.

[0036] Step 5: Enzymatic hydrolysis of RNA into mononucleotides Take 2 μg of total RNA, add 1 μL of nuclease P1 (0.5 U / μL); add 1 μL of buffer, and add water to a final volume of 20 μL; incubate at 37°C for 2 h; add 1 μL of alkaline phosphatase (1 U / μL), and incubate at 37°C for another 1 h; after incubation, heat at 95°C for 5 min to terminate the reaction; centrifuge at 12000 r / min for 10 min, and take 10 μL of the supernatant for LC-MS / MS detection.

[0037] Step 6: Quantitative detection of m by LC-MS / MS (MRM mode) 7 G Chromatographic conditions: Chromatographic column: C18 column (100 mm × 2.1 mm, 1.8 μm); mobile phase: phase A 5 mmol / L ammonium acetate aqueous solution, phase B methanol; elution gradient: 0 → 5 min, 5% → 95% B; Flow rate: 0.2 mL / min; column temperature: 35℃; injection volume: 5 μL.

[0038] Mass spectrometry conditions: Ion source: Electrospray ionization (ESI); Scan mode: Positive ion MRM multiple reaction monitoring; Nebulizer gas: 35 psi; Curtain gas: 20 psi; Collision energy: 15 eV; Quantitative ion pairs: m 7 G: 379.1 → 247.1; G (guanosine): 363.1 → 247.1 Step 7: Data Calculation and Statistics With m 7 Establish a standard curve using G standard sample and calculate m. 7 Absolute content of G; m 7 G relative abundance = m 7 Absolute content of G / Absolute content of total guanosine (rG); The t-test was used for intergroup comparisons, and P < 0.05 was considered statistically significant.

[0039] (4) Experimental results See Figure 1 As shown, among the 50 clinical strains, 25 were drug-resistant strains with m 7 The abundance of G (0.55) was significantly higher than that of the 25 drug-sensitive strains (0.48), while the abundance of m in the 25 drug-resistant strains was significantly higher. 7 The G abundance was 1.458 that of the drug-sensitive strain, and the difference was statistically significant (P=0.046). Furthermore, the m7G modification level in drug-resistant Mycobacterium tuberculosis was positively correlated with the drug-resistant phenotype. (The text abruptly shifts to a different topic: "m7G abundance in Mycobacterium tuberculosis RNA...") 7 The level of G modification can be used as a molecular marker for determining the drug resistance of bacterial strains. Example 2

[0040] We collected Mycobacterium tuberculosis samples from clinical settings, including a drug-sensitive group of 75 samples, an FQ group (resistant to ofloxacin, moxifloxacin, and isoniazid) containing 6 drug-resistant samples, an INH group (resistant to isoniazid and streptomycin) containing 24 drug-resistant samples, an MDR group (resistant to rifampin, rifabutin, streptomycin, isoniazid, para-aminosalicylic acid, ofloxacin, moxifloxacin, and ethambutol) containing 21 drug-resistant samples, an FIR group (resistant to rifampin and rifabutin) containing 9 drug-resistant samples, and an SM group (resistant to streptomycin) containing 15 drug-resistant samples.

[0041] Further analysis of m on RNA across different groups [DS, FQ, INH, MDR, RIF, SM] was performed using the Mann-Whitney U statistical method. 7 Differential analysis of G-modification abundance revealed (e.g.) Figure 2 As shown in the figure, there were significant differences between the FQ group and the sensitive group (DS) (p=0.0228), and there were also significant differences between the SM group and the sensitive group (DS) (p=0.0225), further indicating that m on RNA 7 G modification can serve as a biomarker for tuberculosis resistance (ofloxacin, moxifloxacin, isoniazid, and streptomycin). Example 3

[0042] This embodiment uses AlkAniline-Seq technology to detect Mycobacterium tuberculosis m 7The G-modification site was detected. The strain used in the experiment was Mycobacterium tuberculosis H37Rv. The main reagents included NaOH, HCl, aniline solution, Antarctic Phosphatase, T4 PNK, ligase, and bacterial rRNA removal kit. Library construction was performed using the NEBNext Small RNA Library Prep Set kit, and the sequencing platform was Illumina NovaSeq X Plus. The total RNA extraction method was the same as in Example 2. After extraction, 23S and 16S rRNA were removed using a bacterial rRNA removal kit to enrich mRNA. 2 μg of enriched RNA was added to 50 mM NaOH and lysed at 90 °C for 30 min. After cooling, the RNA was neutralized with HCl, and then antarctic phosphatase was added for dephosphorylation at 37 °C for 1 h. Subsequently, 1 M aniline solution at pH 4.5 was added, and the reaction was carried out at 37 °C for 15 min. After ethanol precipitation and washing, the cleaved RNA was obtained. The RNA was then subjected to T4 PNK phosphorylation, 3' and 5' adapter ligation, reverse transcription, and PCR amplification. PCR amplification was performed using universal primers provided with the NEBNext Small RNA Library Prep Set kit, following the kit instructions. After library quality control, the sequences were sent to Illumina for sequencing in SE50 mode. After sequencing, adapters and low-quality sequences were removed using Cutadapt software. The sequences were then aligned to the H37Rv reference genome using Bowtie2. The stop ratio, normalized cleavage score, and enrichment fold (FC) between the treated and untreated groups were calculated for each locus. High-confidence sequences were selected based on the criteria of Stop Ratio ≥ 0.75, Normalized Cleavage Score ≥ 200, sequencing depth ≥ 5, and FC > 0. 7 G-modified sites.

[0043] Experimental results show that a total of 1110 high-confidence m-values ​​were identified. 7 G-modification sites, these sites are mainly distributed in the coding region of mRNA, and m 7 G modification was positively correlated with protein translation efficiency and protein abundance, and its modification level was significantly increased in drug-resistant strains, demonstrating that m 7 G is involved in regulating the growth, metabolism, stress adaptation, and drug resistance of Mycobacterium tuberculosis; the conclusion shows that AlkAniline-Seq can achieve m 7 G single-base resolution detection, m 7 G can serve as a core biomarker for predicting and studying the mechanisms of tuberculosis drug resistance. Example 4

[0044] Thirty clinical Mycobacterium tuberculosis strains (durability and susceptibility unknown) were collected. Using the standard susceptible strain of Mycobacterium tuberculosis H37Rv as a control, the durability or susceptibility of the clinical Mycobacterium tuberculosis strains was determined through the following steps.

[0045] Experimental reagents: TRIzol, chloroform, isopropanol, 75% ethanol, DEPC, water, Nucleoside Digestion Mix, m 7 G standard; Laboratory equipment: LC-MS / MS system (Xevo TQ), high-speed refrigerated centrifuge, NanoDrop.

[0046] The experimental procedure included: collecting Mycobacterium tuberculosis bacterial culture, centrifuging at 12000 rpm for 10 min to collect bacterial cells, extracting total RNA using the TRIzol method, followed by chloroform extraction, isopropanol precipitation, washing with 75% ethanol, air drying, reconstitution with DEPC water, and setting aside after passing NanoDrop assay; adding 2 μg of total RNA to Nucleoside Digestion Mix enzyme solution, incubating at 37℃ for 1 h, terminating the reaction by heating at 95℃ for 2 min, cooling on ice, centrifuging, and collecting the supernatant for LC-MS / MS (MRM) quantitative detection. Chromatography used a C18 column with gradient elution of 0.1% formic acid aqueous solution and 0.1% formic acid-90% acetonitrile as the mobile phase, and mass spectrometry in ESI+ ion mode. 7 Calculate m for the G(298.1→166.1) and G(268.1→152.1) MRM ion pairs. 7 The relative abundance of G is m 7 The ratio of the peak area of ​​G to the peak area of ​​total guanosine (G); the average m of the drug-sensitive bacterial strain group 7 With a relative abundance of 1.0 as a control, the resistance judgment criteria were set as follows: m of the test strain 7 A relative abundance of G ≥ 1.4 × control indicates a drug-resistant strain, 0.8-1.2 × control indicates a drug-sensitive strain, and 1.2-1.4 indicates a borderline strain, and a retest is recommended.

[0047] The above method was used to perform blind testing on 30 clinical strains, and the isoniazid resistance results determined by the clinically recognized Mycobacterium tuberculosis drug susceptibility test were used as the gold standard to validate the established m 7 The G-modification level detection method was blinded for validation and evaluation. The results showed that among the 30 Mycobacterium tuberculosis strains, 14 were actually drug-resistant and 13 were predicted to be drug-resistant; 16 were susceptible and 14 were predicted to be susceptible.

[0048] The biomarker and prediction method described had a sensitivity of 92.86%, a specificity of 87.50%, an overall concordance rate of 90.00%, a positive predictive value of 86.67%, and a negative predictive value of 93.33%. These experimental results indicate that m 7 G modification level can serve as a rapid, accurate, and stable indicator for assessing drug resistance in Mycobacterium tuberculosis.

[0049] 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. A biomarker for detecting drug resistance in Mycobacterium tuberculosis, characterized in that, The marker is 7-methylguanosine m 7 G modification in drug-resistant Mycobacterium tuberculosis RNA 7 The level of G modification is positively correlated with the degree of drug resistance of Mycobacterium tuberculosis.

2. The drug resistance marker for Mycobacterium tuberculosis according to claim 1, characterized in that, mG modification level in the RNA of the drug-sensitive strain 7 G modification level is the control reference: when the mG modification level in the RNA of the drug-resistant strain is 7 G modification level is ≥ 1.4 times the control reference, it is determined that: drug-resistant Mycobacterium tuberculosis.

3. The drug resistance marker for Mycobacterium tuberculosis according to claim 1, characterized in that, The drug resistance types of the Mycobacterium tuberculosis include: (1) Resistance to first-line anti-tuberculosis drugs, including at least one of isoniazid, rifampin, ethambutol and pyrazinamide; (2) Resistance to second-line anti-tuberculosis drugs, including at least one of fluoroquinolones, bedaquiline, linezolid, clofazimine, streptomycin, kanamycin and amikacin; The drug resistance types include single-drug resistance, multidrug resistance, multidrug-resistant MDR-TB, and extensively drug-resistant XDR-TB.

4. A method for detecting drug resistance in Mycobacterium tuberculosis, characterized in that, Including the following steps: (1) Extract total RNA from the test strain; (2) Detect the modification level of 7-methylguanosine in RNA; (3) Compare the detection results with the m in the RNA of drug-sensitive Mycobacterium tuberculosis. 7 G-level comparison; (4) According to m 7 An elevated G modification level indicates a drug-resistant strain.

5. The method for detecting drug resistance of Mycobacterium tuberculosis according to claim 4, characterized in that, The detection method employed is liquid chromatography-tandem mass spectrometry.

6. The method for detecting drug resistance of Mycobacterium tuberculosis according to claim 4, characterized in that, The detection method includes: Total RNA from the test strain was extracted using the Trizol method or an RNA extraction kit to remove residual genomic DNA; the extracted RNA was digested with a nucleic acid digestion mixture to convert the RNA into mononucleotides in a one-step process; commercially available RNAm was purchased. 7 Using G and rG standards as references, standard solutions were prepared, and a standard detection method was established based on the multi-stage reaction monitoring (MRM) technology of Xevo TQ triple quadrupole mass spectrometry. Among them: rG collected 284.1 m / z precursor ions, 152.1 m / z fragment ions, and retained them for 3.55 min; m 7 G collected precursor ions at 298.1 m / z, fragment ions at 166.1 m / z, with a retention time of 2.46 min; collision energy at 16 V. Based on the established standard MRM procedure, targeted mass spectrometry data acquisition was performed on the mononucleotides obtained from the enzyme digestion of the test strains. By comparing the concentrations of standards, the m-values ​​of RNA molecules in the strains were accurately determined. 7 The G / rG ratio, which is m 7 The relative abundance of G, where a higher relative abundance indicates higher m. 7 The higher the level of G modification.

7. The method for detecting drug resistance of Mycobacterium tuberculosis according to claim 4, characterized in that, In step (4), the m in the RNA of the drug-sensitive strain is used. 7 The G modification level is used as a control baseline: when the RNA of the drug-resistant strain contains m 7 When the G modification level is ≥ 1.4 times that of the control, it is determined to be: drug-resistant Mycobacterium tuberculosis.

8. A method based on m 7 A G-modified detection kit for detecting drug resistance in Mycobacterium tuberculosis, characterized in that... It contains a test kit for detecting 7-methylguanosine, used for the detection of drug resistance in Mycobacterium tuberculosis.

Images