Mycobacterium tuberculosis pas drug resistance molecular marker and application thereof

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a mycobacterium tuberculosis PAS drug resistance molecular marker and application thereof. rv2763c G-70T, G-59T, C-48A mutations determine the drug resistance of bacteria to PAS. The results of the examples show that G-70T, G-59T and C-48A mutations up-regulate the expression level of a target protein coding gene of PAS action, so that Mtb produces drug resistance to PAS. The above-mentioned mycobacterium tuberculosis PAS drug resistance molecular marker provides a new molecular marker for realizing rapid detection of drug resistance of the strain. rv2763c G-70T, G-59T, C-48A mutations determine the drug resistance of bacteria to PAS. The results of the examples show that G-70T, G-59T and C-48A mutations up-regulate the expression level of a target protein coding gene of PAS action, so that Mtb produces drug resistance to PAS. The above-mentioned mycobacterium tuberculosis PAS drug resistance molecular marker provides a new molecular marker for realizing rapid detection of drug resistance of the strain.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a molecular marker of PAS resistance in Mycobacterium tuberculosis and its application. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). The TB epidemic remains severe. Since the introduction of rifampin in 1968, although bedaquiline, delamani, and putomani have been approved as second-line anti-TB drugs, no new first-line anti-TB drugs have been developed. The existing first-line anti-TB treatment regimen (rifampin + isoniazid + pirimethasone + ethambutol for two months, followed by rifampin + isoniazid for four months) has been used globally for over half a century, and drug resistance is inevitable. Traditional first-line drugs such as rifampin and isoniazid face serious challenges from multidrug-resistant and extensively drug-resistant tuberculosis (XDR-TB). Para-aminosalicylic acid (PAS) is one of the earliest clinically used anti-tuberculosis drugs. In recent years, due to the increase in drug-resistant tuberculosis cases, PAS has regained attention as a second-line anti-tuberculosis drug. Although PAS has been used in clinical anti-tuberculosis treatment for many years, its resistance mechanism is still not fully understood; in other words, biomarkers for the molecular diagnosis of PAS resistance need further exploration. Unlike other pathogens, Mtb grows extremely slowly, and traditional bacterial culture-based drug resistance detection methods are too time-consuming (2-3 months), making the need for molecular diagnostics more urgent; the identification of biomarkers is a necessary prerequisite for the molecular diagnosis of Mtb resistance. Summary of the Invention

[0003] The purpose of this invention is to provide a molecular marker for PAS resistance in Mycobacterium tuberculosis and its application; by detecting the presence of PAS in clinical Mtb strains. rv2763c Mutations in the upstream expression regulatory region of the gene, namely G-70T, G-59T, and C-48A, were used to determine the resistance of bacteria to PAS.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of a molecular marker for PAS resistance in Mycobacterium tuberculosis in the detection of PAS resistance in Mycobacterium tuberculosis; the molecular marker is... rv2763c One or more of the following mutations are found in the upstream expression regulatory region of the gene: G-70T, G-59T, and C-48A. rv2763c The reference genome for genes is Mycobacterium tuberculosisH37Rv, GenBank Login Number: AL123456.3. (The following is a separate, unrelated sentence: "The...") rv2763c The C-48A, G-59T, and G-70T mutations in the upstream expression regulatory region of the gene are located at positions 3073657, 3073668, and 3073679 of the genome, respectively.

[0005] Preferably, the application is in the preparation of products for detecting Mycobacterium tuberculosis resistance to PAS.

[0006] A method for detecting drug resistance in Mycobacterium tuberculosis includes: Detection of Mycobacterium tuberculosis in the test rv2763c The gene reaches three mutation sites in the upstream regulatory region: -70, -59, and -48. When the base of the -70 mutation site changes from G to T (G-70T), the base of the -59 mutation site changes from G to T (G-59T), or the base of the -48 mutation site changes from C to A (C-48A), the tested Mycobacterium tuberculosis is resistant to PAS.

[0007] A reagent for detecting the above-mentioned molecular markers, the reagent comprising amplification... rv2763c Primer pairs for upstream expression regulatory regions.

[0008] Preferably, the sequences of the primer pairs are as shown in SEQ ID NO:1-2.

[0009] The present invention also provides a kit for detecting PAS resistance in Mycobacterium tuberculosis, the kit comprising the above-mentioned reagents.

[0010] Preferably, the kit includes one or more of nucleic acid extraction reagents, PCR reagents, and gene-specific primers.

[0011] rv2763c G-70T, G-59T, or C-48A mutations in the upstream expression regulatory region of a gene can increase... rv2763c Applications in gene promoter activity.

[0012] Preferably, the application involves upregulating the gene encoding the target protein of PAS. rv2763c The level of expression.

[0013] Beneficial effects: 1) A new set of Mtb PAS resistance molecular markers of the present invention, specifically: rv2763cMutations in the upstream expression regulatory region of the gene, namely G-70T, G-59T, and C-48A, were identified. Bioinformatics analysis was used to examine the mutations in the upstream expression regulatory region of the rv2763c gene from 6990 clinical Mtb strains, revealing eight mutations. Subsequently, a reporter plasmid containing green fluorescent protein was constructed, and the effects of these eight mutations on… rv2763c The study investigated the effects of three mutations on gene promoter activity, confirming that G-70T, G-59T, and C-48A enhanced promoter activity. Simultaneously, using the Mtb laboratory strain H37Ra as the maternal line, eight mutant strains were constructed, and antimicrobial susceptibility testing confirmed their effectiveness. rv2763c The G-70T, G-59T, and C-48A mutant strains are resistant to PAS.

[0014] 2) G-70T, G-59T, and C-48A mutations upregulate the genes encoding PAS target proteins. rv2763c The expression level of [specific marker] leads to Mtb resistance to PAS. This study of novel biomarkers, based on existing public databases and using bioinformatics methods, identified [specific marker] in clinical Mtb strains. rv2763c Among upstream expression regulatory region mutations, not all mutations lead to Mtb resistance to PAS; some can upregulate expression. rv2763c Mutations in gene expression can lead to PAS resistance, highlighting the crucial role of molecular mechanism research in the identification of molecular markers for drug resistance. Attached Figure Description

[0015] 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.

[0016] Figure 1 Schematic diagram of green fluorescent gene reporter vector construction. A: promoterless GFP negative control reporter vector; B: containing wild-type... rv2763c ( dfrA C: GFP positive control reporter vector for the promoter; C: mutant vector obtained by reverse PCR amplification. dfrA The promoter's gfp report carrier; D: contains only dfrA Negative control vector for promoter; E: containing wild type dfrA Recombinant vector containing promoter and coding region (CDS); F: Recombinant vector obtained by reverse PCR amplification containing... dfrA Coding region (CDS) and mutants dfrA Recombinant vectors for promoters. In this study, pMV261 plasmids linearized by double digestion with AatII and HpaI were used as backbone vectors.

[0017] Figure 2 The expression of the GFP gene in wild-type, control, and mutant strains of *Mycobacterium smegmatis* is shown in Figure A. Growth curves of wild-type, control, and mutant strains of *Mycobacterium smegmatis*; Figures B and C show the expression levels of the GFP gene in wild-type, control, and mutant strains of *Mycobacterium smegmatis*. All data are from triplicate. Ns: No significant difference; **P<0.01, *P<0.05. Detailed Implementation

[0018] This invention provides a molecular marker for PAS resistance in Mycobacterium tuberculosis and its application. To further illustrate this invention, the technical solution provided below is described in detail with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0019] 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.

[0020] 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 legitimate commercial channels and can be prepared according to conventional methods well known to those skilled in the art. The primer and plasmid information used in the embodiments of this invention is as follows: Specific amplification rv2763c Primers for the upstream expression regulatory region sequence of the gene: SEQ ID NO:1 (CGATCCCGATTCCCGGCGCATCATCG) and SEQ ID NO:2 (CGTCACACGCGTCACTCCTTGATTCCGGC).

[0021] Table 1 Primers used for constructing the green fluorescent gene reporter vector and the rv2763c overexpression vector.

[0022] Table 2 Construction of green fluorescent gene reporter vectors and rv2763c The plasmid used for the overexpression vector

[0023] Example 1 Example 1Mtb Clinical strains rv2763c Analysis of mutation distribution in gene expression regulatory regions The whole genome sequences of 6547 clinical isolates of Mycobacterium tuberculosis were retrieved from the National Center for Biotechnology Information (NCBI) database and imported into the bioinformatics software Geneious Prime (version 2022.2.1) for analysis. The H37Rv reference genome (GenBank accession number: AL123456.3) was used as the basis for the analysis. rv2763c Gene (480 bp) and its upstream thyA–dfrA The intergenic spacer region (71 bp) was used as a reference sequence. Sequence alignment of all genomes was performed using the default alignment algorithm of Geneious Prime software to screen for common nucleotide sequences, followed by systematic detection of single nucleotide polymorphisms (SNPs).

[0024] Results: As shown in Table 3, among the 73 clinical isolates rv2763c Eight SNPs were detected in the upstream regulatory region of the gene, among which the G-70T mutation accounted for the highest proportion, reaching 87.67% (64 / 73).

[0025] Table 3. Distribution of mutations in the rv2763c gene expression regulatory region in clinical strains of Mycobacterium tuberculosis.

[0026] Example 2: Mutation Pair in the Regulatory Region rv2763c Effects of gene promoter activity 1. Construction of report carrier Reporter vectors were constructed using the Novizan Rapid Cloning Kit V2, following the kit instructions. First, the pMV261 plasmid was linearized using restriction endonucleases such as AatII and HpaI, and the hsp60 promoter was removed. Primers were designed using the target gene as a template using SnapGene software version 6.0.2; 15–20 bp homologous sequences of the linearized vector were introduced into the 5' ends of the forward and reverse primers, respectively. Mutant, wild-type (WT), and negative control vectors were constructed using different primer combinations synthesized by Qingke Biotechnology (China). The negative control reporter vector used gfp-FP and gfp-RP primers, with the p519ngfp plasmid as the primer. gfp The sequence was used as a template for construction (Figure 1A). When constructing the wild-type reporter vector, the sequence was... rv2763c ( dfrA The upstream 500 bp regulatory region of the gene and gfpGene fusion was performed. Using Mycobacterium tuberculosis H37Ra genomic DNA and p519ngfp plasmid as templates, two sets of primers, wt pro(dfrA)-FP / wt-RP and wt-df.gfp.FP / RP, were used to amplify the upstream regulatory region and... gfp Genes. A 15–20 bp homologous sequence matching the linearized vector and fusion ligation site was added to the 5' end of the primers to ensure successful homologous recombination (Figure 1B). The mutant reporter vector was constructed using a reverse PCR strategy: the wild-type recombinant plasmid was used as a template, and primers introducing site-directed mutations were used to amplify the full-length plasmid. The total recombination reaction system was 10 μL, containing 20 ng of amplified product, 80 ng of the linearized plasmid digested with enzymes, and 5 μL of recombinase buffer (2× CE mix), with enzyme-free water added to the final volume. The reaction mixture was incubated at 50°C for 15 min to complete homologous recombination. The reverse PCR product was digested with DpnI enzyme at 37°C for 1–2 h, followed by gel purification. The recombinant product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing kanamycin, and incubated at 37°C for 24 h. Single colonies were picked for sequencing verification. Correctly identified recombinant vectors were transformed into *Mycobacterium smegmatis* using electroporation for phenotypic analysis. Electroporated bacterial cultures were plated on Middlebrook 7H10 agar plates containing kanamycin and incubated at 37°C for 5 days. After incubation, single colonies were picked and inoculated onto 7H9 liquid medium for colony PCR identification. Detailed information on primers, plasmids, and bacterial strains used is shown in Tables 1 and 2.

[0027] 2. GFP fluorescence detection Mycobacterium smegmatis was cultured to mid-logarithmic growth, then transferred at a 1% inoculum to 5 mL of 7H9 liquid medium supplemented with the appropriate antibiotic, and incubated statically at 37 °C until OD. 600 Approximately 0.6. Take 2 mL of bacterial suspension, centrifuge at 2000×g for 10 min at room temperature to collect bacterial cells; wash the bacterial cells twice with phosphate-buffered saline (PBS), and resuspend in 2 mL of PBS. Add 200 μL of the bacterial suspension to a black 96-well plate with a clear bottom. First, measure the absorbance at 600 nm, then set the excitation wavelength to 488 nm and the emission wavelength to 525 nm to detect the GFP fluorescence intensity. Divide the fluorescence intensity by the OD... 600 The normalized GFP expression level was obtained by calculating the relative fluorescence unit (RFU).

[0028] Results: A recombinant Mycobacterium smegma reporter strain was constructed, which will... dfrA The upstream regulatory region of the gene was fused with a green fluorescent protein (GFP) reporter gene for expression. Growth curve analysis was performed on all reporter strains, including a promoter-free control group (pMV260-). gfp), wild-type promoter recombinant strain (pMV260-PdfrA-) gfp ) and carrying dfrA Strains with different promoter mutants. All strains showed similar growth trends, with no statistically significant differences between groups, indicating that promoter mutations and shuttle plasmids did not cause detectable growth defects, thus ruling out interference from strain growth status on reporter gene expression (Figure 2A). Comparison of GFP expression levels showed that three strains carrying... dfrA The promoter activity of the mutant strains was significantly upregulated compared to the wild type; the other mutations showed downregulated activity or no significant effect. Among the mutant types with upregulated promoter activity, G-70T, G-59T, and C-48A increased promoter activity by approximately 13-fold, 4-fold, and 3-fold, respectively (Figures 2B and 2C).

[0029] The above results indicate that, Mtb Not all of the clinical isolates... dfrA Mutations in the upstream expression regulatory region of a gene can all lead to increased promoter activity, and these mutations need to be identified one by one based on biological information analysis.

[0030] Example 3 rv2763c Mutations in the regulatory region Mtb The impact of PAS resistance 1. In strain H37Ra dfrA Construction of overexpression vectors Using H37Ra genomic DNA as a template, different primers were used to amplify the DNA. dfrA The 500 bp upstream fragment of the gene is related to PdfrA- dfrA The fragment (500 ~ +480 bp region) was successfully used to construct the control vector pMV260-PdfrA and the wild-type vector pMV260-PdfrA- dfrA Each amplified fragment was ligated into the pMV261 vector pretreated with AatII and Hpa. (Targeting...) dfrA Mutant vectors carrying different mutations in the upstream region of the gene were analyzed using reverse PCR with primers containing the mutated sequences from the wild-type vector pMV260-PdfrA-. dfrA The mutant vector was obtained by amplification. The amplified plasmid was digested with DpnI as described above and transformed into *E. coli*, subsequently transformed into *Mycobacterium tuberculosis* strain H37Ra for PAS resistance phenotypic analysis. Detailed information on the primers, plasmids, and strains used is shown in Tables 1 and 2.

[0031] 2. Drug susceptibility testing (DST) The minimum inhibitory concentration (MIC) is defined as the lowest concentration at which an antibiotic can inhibit visible bacterial growth in vitro. This invention determined the inhibitory concentration (MIC) of para-aminosalicylic acid (Sigma-Aldrich) against bacterial growth.Mtb The MIC value of H37Ra was determined. PAS was dissolved in double-distilled water, sterilized by a filter, and then subjected to drug susceptibility testing at final concentration gradients 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. Mtb H37Ra was cultured in 7H9 liquid medium supplemented with OADC until mid-log 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 (CFU / mL) samples were spotted onto 7H10 agar plates containing the corresponding concentration of PAS. The plates were incubated at 37°C for 4 weeks. The MIC value was determined based on the bacterial growth inhibition.

[0032] Result: A carrier was constructed. dfrA Abrupt changes in the upstream regulatory region dfrA The overexpression vector was transformed into strain H37Ra. The efficacy was evaluated by determining the minimum inhibitory concentration (MIC) of PAS. dfrA The effect of upstream expression regulatory region mutations on PAS sensitivity. (See Table 4.) dfrA Mutations in the upstream expression regulatory region that enhance promoter activity can all lead to Mtb Resistance to PAS has developed. Specifically, those carrying... dfrA Overexpressing strains with upstream expression regulatory region mutations G-70T and G-59T exhibited the highest levels of drug resistance compared to wild-type strains, with PAS MIC increased ≥64-fold; the C-48A mutation increased PAS MIC by 32-fold. These results indicate that... rv2763c G-70T, G-59T, and C-48A mutations can be used as... Mtb Molecular markers of PAS resistance.

[0033] Table 4. Minimum inhibitory concentrations of para-aminosalicylic acid against different strains of Mycobacterium tuberculosis.

[0034] As can be seen from the above embodiments: a new set of embodiments of the present invention Mtb PAS resistance molecular markers, specifically rv2763c Mutations in the upstream expression regulatory regions of the gene, namely G-70T, G-59T, and C-48A, were identified. This invention analyzed 6990 strains using bioinformatics methods. Mtb Clinical strains rv2763c Mutations in the upstream expression regulatory region of the gene were investigated, and a total of eight mutations were identified. Subsequently, a reporter plasmid containing green fluorescent protein was constructed, and the effects of these eight mutations on [the gene's expression] were tested. rv2763cThe study investigated the effects of three mutations on gene promoter activity: G-70T, G-59T, and C-48A, which were found to enhance promoter activity. Simultaneously, [the study used]... Mtb Using laboratory strain H37Ra as the maternal line, eight mutant strains were constructed, and antimicrobial susceptibility testing confirmed their mutations. rv2763c The G-70T, G-59T, and C-48A mutant strains are resistant to PAS.

[0035] Accordingly, G-70T, G-59T, and C-48A mutations upregulate the genes encoding PAS target proteins. rv2763c The level of expression leads to Mtb Resistance to PAS has developed. Based on this new set of molecular markers of PAS resistance, extraction... Mtb After obtaining the genome of the clinical strain, specific amplification was performed using primers SEQ ID NO:1 (CGATCCCGATTCCCGGCGCATCATCG) and SEQ ID NO:2 (CGTCACACGCGTCACTCCTTGATTCCGGC). rv2763c The upstream expression regulatory region sequence can be sequenced to detect mutations and determine whether the corresponding clinical strain is resistant to PAS.

[0036] 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. The application of a molecular marker for PAS resistance in Mycobacterium tuberculosis in the detection of PAS resistance in Mycobacterium tuberculosis; said molecular marker is rv2763c One or more of the G-70T, G-59T, and C-48A mutations in the upstream expression regulatory region of the gene.

2. The application according to claim 1, characterized in that, The application is in the preparation of products for detecting PAS resistance in Mycobacterium tuberculosis.

3. A method for detecting drug resistance in Mycobacterium tuberculosis, characterized in that, include: Detection of Mycobacterium tuberculosis in the test rv2763c The gene reaches three mutation sites in the upstream regulatory region: -70, -59, and -48. When the base at the -70 mutation site changes from G to T, the base at the -59 mutation site changes from G to T, or the base at the -48 mutation site changes from C to A, the tested Mycobacterium tuberculosis is resistant to PAS.

4. A reagent for detecting PAS resistance molecular markers in Mycobacterium tuberculosis, said reagent comprising amplification... rv2763c Primer pairs for upstream expression regulatory regions, wherein the molecular marker is rv2763c One or more of the following mutations that reach the regulatory region of the gene: G-70T, G-59T, and C-48A.

5. The reagent according to claim 4, characterized in that, The sequences of the primer pairs are shown in SEQ ID NO:1-2.

6. A kit for detecting PAS resistance in Mycobacterium tuberculosis, characterized in that, The kit includes the reagents described in claim 4 or 5.

7. The reagent kit according to claim 6, characterized in that, The kit includes one or more of the following: nucleic acid extraction reagents, PCR reagents, and gene-specific primers.

8. rv2763c G-70T, G-59T, or C-48A mutations reaching the regulatory region of a gene can increase... rv2763c Applications in gene promoter activity.

9. The application according to claim 8, characterized in that, The application involves upregulating the gene encoding the target protein of PAS. rv2763c The level of expression.

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