Application of rv2763c gene a83t mutation in mycobacterium tuberculosis pas drug resistance detection

Abstract

This invention belongs to the field of molecular biology, specifically relating to a molecular marker for PAS resistance in Mycobacterium tuberculosis and its application. This invention detects the presence of PAS in clinical strains of Mycobacterium tuberculosis. rv2763c The A83T mutation is used to determine resistance to PAS. Examples show that the A83T mutation enhances protein stability, thereby increasing enzyme activity and leading to PAS resistance in Mycobacterium tuberculosis. This invention uses detection... rv2763c The presence of the A83T mutation in Mycobacterium tuberculosis can effectively detect whether Mycobacterium tuberculosis is resistant to PAS, which helps in the analysis of the mechanism of PAS resistance in Mycobacterium tuberculosis and provides a new molecular marker for the rapid detection of drug resistance in this strain.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically involving rv2763c Application of the A83T gene mutation in the detection of PAS resistance in Mycobacterium tuberculosis. Background Technology

[0002] Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis , Mtb Tuberculosis (TB) is an infectious disease caused by infection. According to the World Health Organization, approximately 10 million new cases of TB are diagnosed globally each year, indicating a still serious TB epidemic. The emergence of multidrug-resistant tuberculosis (MDR-TB) has further exacerbated the global TB burden. The increase in MDR-TB severely undermines the effectiveness of existing chemotherapy regimens. Furthermore, due to the extremely slow growth of Mycobacterium tuberculosis, traditional bacterial culture-based methods for detecting drug resistance take 2-3 months to develop. Since determining the drug resistance of the infecting bacteria is a prerequisite for deciding on a treatment strategy, the urgent need for rapid molecular diagnostics is highlighted. Identifying gene mutation markers leading to drug resistance is an essential prerequisite for molecular diagnosis of drug resistance.

[0003] para-aminosalicylic acid ( para β-aminosalicylic acid (PAS) was first used to treat tuberculosis in 1946 and has been widely used for decades. As early as 1952, PAS was used in combination with streptomycin to prevent the development of drug resistance. However, after the discovery of more effective drugs such as isoniazid and rifampin, PAS was gradually discontinued due to its relatively unfavorable side effects. But with the resurgence of multidrug-resistant tuberculosis in the 1990s, PAS was reintroduced as a second-line anti-tuberculosis drug. Currently, PAS is frequently included as a group C drug in the intensive treatment phase of multidrug-resistant tuberculosis treatment regimens.

[0004] Known PAS resistance-related genes, such as folC , thyA , thyX , ribD and rv2763c ( dfrA / folA It has been shown to encode enzymes involved in folate metabolism in Mycobacterium tuberculosis, but its drug resistance mechanism is not yet fully understood, and the markers for molecular diagnosis of drug resistance need to be further explored. Summary of the Invention

[0005] The purpose of this invention is to provide rv2763cThe application of the A83T gene mutation in the detection of PAS resistance in Mycobacterium tuberculosis, by detecting the presence of PAS in clinical strains of Mycobacterium tuberculosis. rv2763c The A83T mutation determines bacterial resistance to PAS.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides: rv2763c Application of the A83T gene mutation in the detection of PAS resistance in Mycobacterium tuberculosis.

[0007] The rv2763c The A83T gene mutation is rv2763c An A>T mutation occurred at position 83 of the coding gene. rv2763c The reference genome for genes is Mycobacterium tuberculosis H37Rv, GenBank Login Number: AL123456.3. (The following is a separate, unrelated sentence: "The...") rv2763c The A83T mutation is located at position 3073527 of the genome.

[0008] Preferably, the application is as follows: rv2763c Application of the A83T gene mutation in the preparation of products for detecting or predicting Mycobacterium tuberculosis resistance to PAS.

[0009] The present invention also provides a method for detecting drug resistance in Mycobacterium tuberculosis, comprising: Detection of Mycobacterium tuberculosis in the test rv2763c The A83T mutation site in the gene indicates that the tested Mycobacterium tuberculosis strain is resistant to PAS when the base at this mutation site is detected to be T.

[0010] Preferably, the detection method is sequencing, PCR, hybridization, or mass spectrometry.

[0011] A reagent for detecting PAS resistance molecular markers in Mycobacterium tuberculosis, the reagent comprising amplification... rv2763c The primer pair for the gene, the molecular marker being rv2763c A83T mutation in the coding region of the gene.

[0012] Preferably, the sequences of the primer pairs are as shown in SEQ ID NO:1 to 4.

[0013] A kit for detecting PAS resistance in Mycobacterium tuberculosis, the kit comprising the above-described reagents.

[0014] Preferably, the kit includes one or more of nucleic acid extraction reagents, PCR reagents, and gene-specific primers.

[0015] rv2763c Application of gene A83T mutation in enhancing the stability of its encoded protein and the activity of its encoded protease.

[0016] A strain resistant to PAS, said strain being a carrier rv2763c Gene A83T mutation.

[0017] Beneficial effects: This invention discloses for the first time a method that can mediate PAS resistance. rv2763c The A83T mutation site in the gene. This invention has performed cloning verification targeting the above SNP mutations, and will clone genes containing the A83T mutation. rv2763c Gene( rv2763c A83T The SNP mutation was cloned into the original strain without the mutation site, and the original strain's MIC value for PAS increased eightfold (from 0.08 μg / mL to 0.64 μg / mL), confirming that this SNP mutation can mediate PAS resistance. Therefore, this invention, through detection... rv2763c The presence of the A83T mutation in Mycobacterium tuberculosis can effectively detect whether Mycobacterium tuberculosis is resistant to PAS, which helps in the analysis of new mechanisms of PAS resistance in Mycobacterium tuberculosis and provides a new molecular marker for the rapid detection of drug resistance in this strain. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 For those with A83T mutation rv2763c Gene( rv2763c A83T Schematic diagram of the construction of overexpression vector and control vector. A, carrying wild type rv2763c The gene vector served as a negative control; B, carrying the A83T mutation. rv2763c Gene expression vector; AatII and HpaI are restriction endonucleases on pMV261, light blue and dark blue represent primer sequence overlap regions, and red represents regions containing the A83T mutation site. Detailed Implementation

[0020] This invention provides rv2763cApplication of the A83T gene mutation in the detection of PAS resistance in Mycobacterium tuberculosis. To further illustrate this invention, the technical solutions provided by this invention are described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0021] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0022] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0023] This invention constructs rv2763c A83T The primers used for the overexpression vector and the control vector are shown in Table 1: Table 1 Construction rv2763c A83T Primers used for overexpression vector and control vector

[0024] rv2763c A83T Schematic diagrams of the construction of mutant overexpression vectors and control vectors are shown below. Figure 1 As shown in Table 2, the list of carriers involved in this invention is as follows:

[0025] Table 2 is a list of carriers involved in this invention.

[0026] Example 1: Clinical strains rv2763c Analysis of mutation distribution in gene coding regions

[0027] Seven multidrug-resistant Mycobacterium tuberculosis (MTB) clinical isolates were collected from the Chongqing Public Health Medical Treatment Center. Drug susceptibility testing showed that all seven were resistant to PAS. Genomic DNA was extracted from these seven resistant isolates using the proteinase K method and sent to Shanghai Bio-Tech Co., Ltd. for whole-genome sequencing. The sequencing results were uploaded to the National Center for Biotechnology Information (NCBI) database (accession number: PRJNA774929). Simultaneously, a search of the NCBI database yielded the whole-genome sequences of 6547 MTB clinical isolates. All the above genomic data were imported into the bioinformatics software Geneious Prime (version 2022.2.1) for analysis. The H37Rv reference genome (GenBank accession number: AL123456.3) was used for the analysis. rv2763c The gene (480 bp) was used as a reference sequence. The Geneious Prime software's default alignment algorithm was used to align all genomes and screen for common nucleotide sequences. Subsequently, single nucleotide polymorphisms (SNPs) were systematically detected.

[0028] Results: Search results show rv2763c The coding region contained 29 SNPs, including 20 missense mutations, 5 synonymous mutations, 3 frameshift mutations, and 1 termination mutation. Among these, W142G was the most common mutation type (27.27%), while all other mutations were observed in no more than two strains each; it was found in strain E847 collected by the Chongqing Public Health Medical Treatment Center. rv2763c The gene has an A83T mutation at position 83.

[0029] Example 2: Predicting the effect of coding region mutations on the thermal stability of Rv2763c protein using bioinformatics methods

[0030] 1. Query the crystal structure of Rv2763c protein (PDB ID: 6nnd) from the PDB database (https: / / www.rcsb.org / ) and download the result file (pdb format).

[0031] 2. Input the above results file and the single-point missense mutation information of the Rv2763c protein into the PremPS website (https: / / lilab.jysw.suda.edu.cn / researc-h / PremPS / ) to obtain the ΔΔG value of each mutant amino acid. ΔΔG is defined as: the mutant ΔG value minus the wild-type ΔG value. A negative value indicates that the mutant protein is more stable than the wild-type protein, while a positive value indicates that the mutant protein is less stable than the wild-type protein.

[0032] Results: As shown in Table 3, most mutations had positive ΔΔG values, while the Q28L mutation (corresponding to the A83T mutation in the coding region) had a negative ΔΔG value, indicating that, based on bioinformatics prediction, most... rv2763c Mutations in the coding region decrease the thermal stability of proteins, while the Q28L mutation increases stability. Furthermore, decreased thermal stability is often associated with decreased enzyme activity, while increased thermal stability may enhance enzyme activity.

[0033] Table 3 Thermal stability analysis of Rv2763c mutant protein

[0034] Example 3 Mycobacterium tuberculosis rv2763c The impact of coding region mutations on PAS resistance

[0035] 1. In strain H37Ra rv2763c Construction of overexpression vectors

[0036] Using SnapGene (Version 6.0.2) software, the strain of Mycobacterium tuberculosis H37Ra was analyzed. rv2763c The 500 bp upstream fragment of the gene and its 480 bp coding gene (P) rv2763c - rv2763c The upstream and downstream primers were designed using the template, with the 5' end of the upstream primer p-dfrA-FP introduced into the pMV261 vector. Aat The 21 bp homologous sequence upstream of the II restriction site; the 5' end of the downstream primer dfrA-RP is introduced into the pMV261 vector. Hpa A 21 bp homologous sequence downstream of the I restriction site. This allows the PCR amplification product to contain... rv2763c Upstream promoter sequence of the gene (P) rv2763c )and rv2763c The region encoding the gene (-500 ~ +480). Use... Aat II and Hpa The pMV261 plasmid was double-digested with restriction endonucleases at 37°C for 1 h to remove its hsp60 promoter region, yielding a linear pMV261 plasmid (the pMV261 vector with the hsp60 promoter region removed was named pMV260). Double digestion was verified by gel electrophoresis, and the linear plasmid was purified using a gel extraction kit. The plasmid was then purified using Trelief... TMExpression vectors were constructed using the SoSoo cloning kit. The ligation mixture was prepared according to the kit instructions (10 ng pMV261 digestion product, 30 ng PCR amplification product, 5 μL 2×SoSoo mix, and ddH2O to a final volume of 10 μL). After gentle mixing, the mixture was incubated at 50°C for 15 min, then cooled on ice for 5 min. The ligation product was then cloned into *E. coli* DH5α via electroporation. Wild-type cloning was obtained after verification. rv2763c Overexpression vector pMV260-P rv2763c - rv2763c .

[0037] against rv2763c The overexpression vector of the mutant gene was generated using reverse PCR with primers containing the mutant sequence from the wild-type vector pMV260-P. rv2763c - rv2763c The overexpression vector containing the mutation was obtained through amplification. The amplified product was digested with DpnI and transformed into *E. coli*. After verification, the mutant strain was obtained. rv2763c Overexpression vector pMV260-P rv2763c - rv2763c A83T pMV260-P rv2763c - rv2763c G95C and pMV260-P rv2763c - rv2763c T424G .

[0038] The above recombinant plasmid was cloned into Mycobacterium tuberculosis H37Ra by electroporation to obtain the recombinant strain H37RapMV260-P rv2763c - rv2763c H37Ra pMV260-P rv2763c - rv2763c A83T H37Ra pMV260-P rv2763c - rv2763c G95C , and H37Ra pMV260-P rv2763c - rv2763c T424G .

[0039] 2. Drug susceptibility testing (DST)

[0040] The minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antibiotic that can inhibit visible bacterial growth in vitro. In this example, the MIC of PAS (Sigma-Aldrich) against Mycobacterium tuberculosis H37Ra was determined. PAS was dissolved in double-distilled water, filtered, and then subjected to a final concentration gradient of 0, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56, and 5.12 μg / mL for drug susceptibility testing. Each recombinant strain was cultured in 7H9 liquid medium supplemented with OADC to mid-logarithmic growth, and the bacterial concentration was adjusted to approximately 10. 7 CFU / mL, then diluted 100 times with fresh culture medium. Take 10 μL of bacterial suspension (approximately 10... 5 The sample (CFU / mL) was spotted onto a 7H10 agar plate containing the corresponding concentration of PAS and then incubated at 37°C for 4 weeks. The MIC value was determined based on the bacterial growth inhibition.

[0041] Results: Wild-type was constructed. rv2763c Gene and coding region mutations rv2763c The gene overexpression vector was generated and transformed into the H37Ra strain. The MIC of PAS was evaluated. rv2763c The impact of coding region mutations on PAS susceptibility. As shown in Table 4, the A83T mutation can lead to PAS resistance in Mycobacterium tuberculosis; while G95C and T424G cannot. Specifically, those carrying... rv2763c A83T The mutant overexpression strain exhibited the highest level of drug resistance compared to the wild type, with an 8-fold increase in MIC. These results indicate that... rv2763c The A83T mutation in the gene can serve as a molecular marker for detecting PAS resistance in Mycobacterium tuberculosis.

[0042] Table 4. Minimum inhibitory concentration of PAS against Mycobacterium tuberculosis strains with overexpression of mutant genes.

[0043] As can be seen from the above embodiments, a novel molecular marker for PAS resistance in Mycobacterium tuberculosis according to the present invention is specifically as follows: rv2763c The A83T mutation in the gene coding region. The inventors analyzed 6547 clinical strains of Mycobacterium tuberculosis using whole-genome sequencing and bioinformatics methods. rv2763cMutations in the coding region were investigated, identifying 16 single-point missense mutations. Bioinformatics methods were then used to analyze the impact of these 16 mutations on the thermal stability of the Rv2763c protein, predicting that the Q28L amino acid sequence mutation (corresponding to the A83T mutation in the coding region) would enhance protein thermal stability. Simultaneously, three mutant overexpression strains were constructed using Mycobacterium tuberculosis H37Ra as the maternal strain, and antimicrobial susceptibility testing confirmed the mutations. rv2763c The A83T mutant strain is resistant to PAS.

[0044] Accordingly, the A83T mutation enhances protein stability, thereby increasing enzyme activity and leading to resistance to PAS in Mycobacterium tuberculosis. Based on this novel molecular marker of PAS resistance, the genome of a clinical strain of Mycobacterium tuberculosis was extracted and specifically amplified. rv2763c The coding region sequence can be sequenced to detect mutations and determine whether the corresponding clinical strain is resistant to PAS.

[0045] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. rv2763c Application of the A83T gene mutation in the detection of PAS resistance in Mycobacterium tuberculosis.

2. The application according to claim 1, characterized in that, The application is rv2763c Application of the A83T gene mutation in the preparation of products for detecting or predicting Mycobacterium tuberculosis resistance to PAS.

3. A method for detecting drug resistance in Mycobacterium tuberculosis, characterized in that, include: Detection of Mycobacterium tuberculosis in the test rv2763c The A83T mutation site in the gene indicates that the tested Mycobacterium tuberculosis strain is resistant to PAS when the base at this mutation site is detected to be T.

4. The method according to claim 3, characterized in that, The detection method is sequencing, PCR, hybridization, or mass spectrometry.

5. A reagent for detecting PAS resistance molecular markers in Mycobacterium tuberculosis, characterized in that, The reagents include amplification. rv2763c The primer pair for the gene, the molecular marker being rv2763c A83T mutation in the coding region of the gene.

6. The reagent according to claim 5, characterized in that, The sequences of the primer pairs are shown in SEQ ID NO:1-4.

7. A kit for detecting PAS resistance in Mycobacterium tuberculosis, characterized in that, The kit includes: capabilities for detecting... rv2763c A reagent for the base type of the A83T mutation site in the gene.

8. The reagent kit according to claim 7, characterized in that, The reagent includes the reagent described in claim 5 or 6.

9. rv2763c Application of gene A83T mutation in enhancing the stability of its encoded protein and the activity of its encoded protease.

10. A strain resistant to PAS, characterized in that, The strain is a carrier. rv2763c Gene A83T mutation.

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