Primer group for detecting drug-resistant gene loci of mycobacterium tuberculosis and preparation method of primer group

Abstract

The invention discloses a primer group for detecting drug-resistant gene loci of mycobacterium tuberculosis and a preparation method of the primer group, and particularly relates to the technical field of gene mutation detection, the primer group comprises amplification primers and single-base extension primers of various mycobacterium tuberculosis drug-resistant genes, a plurality of loci of genes such as rpoB, inhA, katG, eis, rrs, gyrB and gyrA are covered, and by means of primer design, synthesis and proportioning, the drug-resistant gene loci of the mycobacterium tuberculosis can be detected. The method comprises the steps of sample DNA extraction, PCR amplification, SAP enzyme digestion, single base extension reaction, product analysis and the like, so that the detection of the drug-resistant gene locus of the mycobacterium tuberculosis is realized. The kit can be used for accurately typing the drug resistance-related point mutation of the mycobacterium tuberculosis, and has the advantages of high accuracy, high flux, good repeatability, automation and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of gene mutation detection, and more specifically, to a primer set for detecting drug-resistant gene sites of Mycobacterium tuberculosis and a preparation method thereof. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, which seriously endangers human health. Globally, there are a large number of tuberculosis patients, and drug resistance has become a major challenge to treatment.

[0003] At present, the detection methods of Mycobacterium tuberculosis drug resistance mainly include traditional culture drug sensitivity test, Sanger sequencing technology and second-generation sequencing technology. Traditional culture drug sensitivity test has high credibility, but the culture detection cycle is long and cannot provide timely treatment basis for patients; Sanger sequencing technology has high accuracy, but low detection efficiency and high cost, which is difficult to meet the needs of large-scale detection; second-generation sequencing technology has high sensitivity, but high cost, which limits its wide application in clinical practice.

[0004] The existing technologies have certain limitations in detecting drug-resistant gene loci of Mycobacterium tuberculosis, such as incomplete detection loci, unreasonable primer design, insufficient sensitivity and specificity of detection methods, etc. Some technologies involve the detection of drug-resistant genes of Mycobacterium tuberculosis, but they often only focus on some gene loci and fail to cover all important drug-resistant gene loci; unreasonable primer design may lead to low amplification efficiency and poor specificity, affecting the accuracy of the test results; insufficient sensitivity and specificity of the detection method may lead to missed detection and false detection, bringing risks to clinical treatment.

[0005] Therefore, a primer set for detecting drug-resistant gene loci of Mycobacterium tuberculosis and a preparation method thereof are proposed. Summary of the invention

[0006] In order to overcome the above defects of the prior art, the present invention provides a primer set for detecting drug-resistant gene sites of Mycobacterium tuberculosis and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions: a primer set for detecting drug-resistant gene loci of Mycobacterium tuberculosis, comprising core gene loci primers and other loci primers;

[0008] The core gene locus primers include:

[0009] rpoB gene primers, including but not limited to 533 site (rpoB-533), 511 site (rpoB-511), 513 site (rpoB-513-1), 482 site (rpoB-482), 522 site (rpoB-522), 526 site (rpoB-526-1, rpoB-526-2), 531 site (rpoB-531-1, rpoB-531-2, rpoB-531-3), 516 site (rpoB-516), etc., and corresponding amplification primers and extension primers, the sequences of the extension primers are shown in SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQID NO:71, etc.;

[0010] inhA gene primers, including amplification primers and extension primers for sites such as (-15) site (inhA-15) and (-8) site (inhA-8);

[0011] katG gene primers, including amplification primers and extension primers for sites such as site 315 (katG-315);

[0012] eis gene primers, including amplification primers and extension primers at the (-37) site (eis-promoter-37), (-10) site (eis-promoter-10), (-14) site (eis-promoter-14) and other sites in the promoter region;

[0013] rrs gene primers, including amplification primers and extension primers for sites 1321 (rrs-1321), 1401 (rrs-1401), and 1300 (rrs-1300);

[0014] gyrB gene primers, including amplification primers and extension primers for sites 538 (gyrB-538), 500 (gyrB-500), and 540 (gyrB-540);

[0015] gyrA gene primers, including amplification primers and extension primers at positions 74 (gyrA-74), 90 (gyrA-90), 94 (gyrA-94), and 91 (gyrA-91);

[0016] The other site primers include other site primers of the rpoB gene, such as primers of sites such as F505V, S512T, and D626D;

[0017] Primers for other sites of the gyrB gene, such as primers for sites such as N538I, G509C, and T539N;

[0018] Primers for other sites of the eis gene, such as primers for C(-12)T, C(-2)T, G37T and other sites.

[0019] Preferably, the primer component consists of 3 Wells, specifically as follows:

[0020] Well1: rpoB-533, gyrA-90, rpoB-511, rpoB-513-1, rpoB-482, rrs-1321, gyrB-538, rrs-14 01. gyrA-74, rrs-1300, rpoB-522, katG-315, rpoB-526-1, gyrA-94, gyrB-500, rpoB-526-2;

[0021] Well2: rpoB-531-1, rpoB-531-2, rpoB-513-2, rpoB-572, inhA-15, gyrB-540, rpoB-522, gyrA-94;

[0022] Well3: rpoB-513-3, rpoB-516, eis-promoter-37, eis-promoter-10, eis-promoter-14, rpoB-531-3, rpoB-516, inhA-8, gyrA-91.

[0023] Preferably, the primer has 15 or more bases at the 3' end that completely match the target gene sequence, and a universal tag sequence of 10 bases (ACGTTGGATG) at the 5' end.

[0024] A method for preparing a primer set comprises the following steps:

[0025] Primer design: Based on the latest research results of Mycobacterium tuberculosis resistance genes and known resistance sites, combined with bioinformatics analysis, amplification primers and single-base extension primers for each gene site are designed, taking into account factors such as primer specificity, amplification efficiency, and annealing temperature. Primers are screened and optimized using computer software to ensure that primers can accurately amplify and detect target gene sites. Sequence alignment and homology analysis are performed on the designed primers to avoid cross-hybridization and nonspecific amplification between primers;

[0026] Primer synthesis: The designed primers are synthesized by chemical synthesis method, and the synthesized primers are subjected to high performance liquid chromatography (HPLC) or other appropriate purification methods to improve the purity and quality of the primers. The concentration and quality of the synthesized primers are measured to ensure that the concentration of the primers meets the requirements of subsequent experiments and are free of impurities and degradation products;

[0027] Primer ratio: According to the detection requirements and experimental experience of the MassARRAY nucleic acid mass spectrometry platform, the single-base extension primers are accurately ratioed to ensure that the amplification efficiency of each single-base extension primer in the single-hole reaction system is consistent and that the products of different molecular masses after extension can be accurately detected in the flying nucleic acid mass spectrometry;

[0028] The specific ratio is as follows:

[0029] Well1: rpoB-533 corresponds to primer SEQ ID NO:67 at a ratio of 2.64 μl, gyrA-90 corresponds to primer SEQ ID NO:68 at a ratio of 2.89 μl, rpoB-511 corresponds to primer SEQ ID NO:69 at a ratio of 3.53 μl, rpoB-513-1 corresponds to primer SEQ ID NO:70 at a ratio of 4.41 μl, rpoB-482 corresponds to primer SEQ ID NO:71 at a ratio of 4.91 μl, rrs-1321 corresponds to primer SEQ ID NO:72 at a ratio of 5.69 μl, gyrB-538 corresponds to primer SEQ ID NO:73 at a ratio of 5.96 μl, rrs-1401 corresponds to primer SEQ ID NO:74 at a ratio of 6.59 μl, and gyrA-74 corresponds to primer SEQ ID NO:75 at a ratio of 5.70 μl. The ratio of primer SEQ ID NO:75 is 6.95 μl, the ratio of primer SEQ ID NO:76 corresponding to rrs-1300 is 6.99 μl, the ratio of primer SEQ ID NO:77 corresponding to rpoB-522 is 7.01 μl, the ratio of primer SEQ ID NO:78 corresponding to katG-315 is 7.44 μl, the ratio of primer SEQ ID NO:79 corresponding to rpoB-526-1 is 7.62 μl, the ratio of primer SEQ ID NO:80 corresponding to gyrA-94 is 7.71 μl, the ratio of primer SEQ ID NO:81 corresponding to gyrB-500 is 7.97 μl, the ratio of primer SEQ ID NO:82 corresponding to rpoB-526-2 is 8.23 ​​μl, and the water is added to 398.2 μl;

[0030] Well2: rpoB-531-1 corresponding to primer SEQ ID NO:83 is 2.71 μl, rpoB-531-2 corresponding to primer SEQ ID NO:84 is 3.65 μl, rpoB-513-2 corresponding to primer SEQ ID NO:85 is 4.41 μl, rpoB-572 corresponding to primer SEQ ID NO:86 is 4.52 μl, inhA-15 corresponding to primer SEQ ID NO:87 is 5.89 μl, gyrB-540 corresponding to primer SEQ ID NO:88 is 6.45 μl, rpoB-522 corresponding to primer SEQ ID NO:89 is 5.10 μl, gyrA-94 corresponding to primer SEQ ID NO:90 is 6.37 μl, make up to 461 μl with water;

[0031] Well3: rpoB-513-3 corresponding to primer SEQ ID NO:91 was 4.41 μl, rpoB-516 corresponding to primer SEQ ID NO:92 was 5.97 μl, eis-promoter-37 corresponding to primer SEQ ID NO:93 was 8.02 μl, eis-promoter-10 corresponding to primer SEQ ID NO:94 was 8.13 μl, eis-promoter-14 corresponding to primer SEQ ID NO:95 was 9.48 μl, rpoB-531-3 corresponding to primer SEQ ID NO:96 was 3.51 μl, rpoB-516 corresponding to primer SEQ ID NO:97 was 5.32 μl, inhA-8 corresponding to primer SEQ ID NO:98 was 6.28 μl, gyrA-91 corresponding to primer SEQ ID NO:99 was 7.84 μl, The ratio of NO:99 is 6.63μl, add water to 442μl.

[0032] A method for detecting drug-resistant gene loci of Mycobacterium tuberculosis using the primer set described above, characterized in that it comprises the following steps:

[0033] Sample preparation: Collect clinical samples, such as sputum, blood, urine, etc. The samples should be as fresh as possible to avoid contamination and degradation. Then use conventional DNA extraction methods, such as phenol-chloroform method, kit extraction method, etc., to extract the DNA of Mycobacterium tuberculosis from the samples. Determine the concentration and purity of the extracted DNA to ensure that the DNA quality meets the detection requirements, such as the DNA concentration should be greater than 50ng / μl, and the A260 / A280 ratio should be between 1.8-2.0;

[0034] PCR amplification: Prepare the PCR reaction system, mix the extracted DNA, primers, PCR enzyme, buffer, dNTP, etc. in a certain proportion to ensure the accuracy and stability of the reaction system, and perform PCR amplification reaction. The reaction conditions are: denaturation at 95°C for 2 minutes, followed by 45 cycles, each cycle including denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, and finally extension at 72°C for 5 minutes. During the amplification process, attention should be paid to controlling the reaction temperature and time to avoid abnormalities in the PCR reaction;

[0035] SAP enzyme digestion: SAP enzyme digestion is performed on the PCR amplification product to remove excess dNTPs in the reaction and reduce interference with the extension reaction. The SAP reaction system is prepared by mixing the PCR amplification product, SAP enzyme, buffer, etc. in a certain proportion and performing SAP enzyme digestion reaction. The reaction conditions are: digestion at 37°C for 40 minutes, inactivation at 85°C for 5 minutes, and then storage at 4°C. After the digestion reaction is completed, the subsequent extension reaction should be carried out in time to avoid DNA degradation;

[0036] Single base extension reaction: Use single base extension primer to perform single base extension reaction on the digestion product to obtain extension product, prepare iPLEX extension reaction system, mix the SAP digestion product, iPLEX enzyme, extension primer, buffer, etc. in a certain proportion, and perform single base extension reaction. The reaction conditions are: denaturation at 94℃ for 30 seconds, followed by 40 cycles, each cycle includes denaturation at 94℃ for 5 seconds, annealing at 52℃ for 5 seconds, extension at 80℃ for 5 seconds, and finally extension at 72℃ for 3 minutes. During the extension reaction, the reaction temperature and time should be strictly controlled to ensure the accuracy of the extension reaction.

[0037] Product analysis: Desalting and purifying the extension products to remove residual salt ions in the reaction and improve the accuracy of mass spectrometry analysis. Using nucleic acid mass spectrometry analysis software to type the sites and determine whether there are mutations. Compare the test results of the samples with known drug resistance standards to analyze the type and degree of drug resistance of the samples. Record and analyze the test results and generate a test report to provide a reference for clinical treatment.

[0038] Preferably, the volume ratio of the PCR amplification reaction solution, the SAP digestion reaction solution and the iPLEX reaction solution is 5:2:2.

[0039] An application of the primer set in preparing a kit for detecting drug resistance of Mycobacterium tuberculosis.

[0040] A Mycobacterium tuberculosis drug resistance detection kit, comprising the above-mentioned primer set and a single-base extension primer prepared according to the primer ratio scheme; the kit consists of:

[0041] Primer reagents: Contains various amplification primers and single-base extension primers, which are packed in different tubes and marked with primer names and sequence information;

[0042] PCR reaction reagents: including 10xPCRBuffer, 25mMMgCl2, 25mMdNTPMix, PCREnzyme, etc., prepared in a certain proportion and packaged in the kit;

[0043] SAP enzyme reaction reagents: including NanopureWater, SAPBuffer, SAPEnzyme, etc., prepared in a certain proportion and packaged in the kit;

[0044] iPLEX reaction reagents: including iPLEX Buffer, iPLEX Termination Mix, Extend Primer Mix, iPLEX Enzyme, etc., prepared in a certain proportion and packaged in the kit;

[0045] Other reagents: such as resin, chip boards, standards, etc., are prepared according to experimental needs.

[0046] Method for using the kit

[0047] Sample processing: Collect and process samples according to the instructions to extract DNA of Mycobacterium tuberculosis;

[0048] PCR amplification: Mix the extracted DNA, primer reagents, and PCR reaction reagents in a certain proportion to perform PCR amplification reaction;

[0049] SAP enzyme digestion: The PCR amplification product was digested with SAP enzyme;

[0050] Single base extension reaction: Use single base extension primer to perform single base extension reaction on the digestion product;

[0051] Product analysis: The extension products were desalted and purified, and then detected and analyzed using nucleic acid mass spectrometry software.

[0052] An application of the primer set or kit in the detection of drug resistance of Mycobacterium tuberculosis includes but is not limited to the following aspects:

[0053] Clinical diagnosis: used to detect drug resistance of Mycobacterium tuberculosis in clinical samples to provide a basis for clinical treatment;

[0054] Drug research and development: assist drug research and development companies in drug screening and drug resistance mechanism research, and develop new anti-tuberculosis drugs;

[0055] Public health monitoring: used for monitoring, prevention and control of tuberculosis, understanding the drug resistance of Mycobacterium tuberculosis, and formulating reasonable prevention and control strategies.

[0056] Technical effects and advantages of the present invention:

[0057] 1. The primer set of the present invention covers multiple sites of various Mycobacterium tuberculosis resistance genes, including some newly discovered sites and important resistance-related sites, and can comprehensively detect the drug resistance of Mycobacterium tuberculosis, providing more comprehensive information for clinical treatment.

[0058] 2. Through careful design and optimization of primers, the specific binding of primers to target gene sites is ensured, thereby improving amplification efficiency and detection accuracy.

[0059] 3. Accurately proportion the single-base extension primers to ensure consistent amplification efficiency of each primer in the single-well reaction system, reducing detection errors caused by primer differences.

[0060] 4. Strictly control the reaction conditions of PCR amplification, SAP enzyme digestion and single base extension reaction to ensure the stability and repeatability of the detection process and improve the reliability of the test results.

[0061] 5. It can accurately detect the drug-resistant gene sites of Mycobacterium tuberculosis, provide clinical physicians with personalized medication references, achieve precise treatment, and improve treatment effects, thereby enabling early detection of the drug resistance of Mycobacterium tuberculosis, helping to adjust treatment plans in a timely manner, avoiding the spread of drug-resistant strains, and reducing the harm of tuberculosis.

[0062] 6. The primer set is divided into 3 Wells, and the primers are reasonably allocated according to the characteristics of different sites and detection requirements, which improves the efficiency and accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figures 1-6 are result diagrams of some samples in Example 2, where:

[0064] Figure 1A :Mass spectrometry peak of sample S234 rpoB 526 site;

[0065] Figure 1B :Generation sequencing verification peak diagram of sample S234 rpoB 526 site;

[0066] Figure 2A :Mass spectrum peak of sample y11 rpoB 482 site;

[0067] Figure 2B :Generation sequencing verification peak diagram of sample y11 rpoB 482 site;

[0068] Figure 3A :Mass spectrum peak of sample y54 gyrA 90 site;

[0069] Figure 3B :Generation sequencing verification peak diagram of sample y54 gyrA 90 site;

[0070] Figure 4A :Mass spectrometry peak of sample y81 rpoB 572 site;

[0071] Figure 4B :Generation sequencing verification peak diagram of sample y81 rpoB 572 site;

[0072] Figure 5A :Mass spectrum peak of sample y126 gyrA 94 site;

[0073] Figure 5B :Generation sequencing verification peak diagram of sample y126 gyrA 94 site;

[0074] Fig. 6A :Mass spectrum peak diagram of sample y252 rrs 1321 site;

[0075] Figure 6B :Generation sequencing verification peak diagram of sample y252 rrs 1321 site. DETAILED DESCRIPTION

[0076] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0077] Embodiment 1

[0078] Experimental Materials

[0079] Primer set

[0080] A primer set was synthesized, including amplification primers and single base extension primers for genes such as rpoB, inhA, katG, eis, rrs, gyrB and gyrA, as shown in Table 2. For example, the sequence is as follows:

[0081] rpoB-533: upstream primer (SEQ ID NO: 1), downstream primer (SEQ ID NO: 2), extension primer (SEQ ID NO: 67);

[0082] gyrA-90: upstream primer (SEQ ID NO: 3), downstream primer (SEQ ID NO: 4), extension primer (SEQ ID NO: 68);

[0083] rpoB-511: upstream primer (SEQ ID NO: 5), downstream primer (SEQ ID NO: 6), extension primer (SEQ ID NO: 69);

[0084] In order to ensure that the amplification efficiency of each single-base extension primer in the single-well reaction system is consistent and the products of different molecular masses after extension can be accurately detected in the flying nucleic acid mass spectrometry, the single-base extension primers (starting concentration 500 μM) are combined according to the following ratio: (reaction wells 1, 2, 3), and the primers are purified by ePAGE after synthesis to ensure that the purity meets the requirements.

[0085]

[0086]

[0087]

[0088]

[0089] Samples: Sputum samples from 50 tuberculosis patients were collected clinically and stored in a -80℃ refrigerator immediately after collection.

[0090] Reagents

[0091] PCR reagents

[0092] 10xPCRBuffer (containing 20mM MgCl2);

[0093] 25mM MgCl2;

[0094] 25mM dNTPMix;

[0095] Taq DNA polymerase (5 U / μL).

[0096] SAP enzyme reagent

[0097] NanopureWater;

[0098] SAPBuffer;

[0099] SAPEnzyme (1.7U / μL).

[0100] iPLEX Reagents

[0101] iPLEXBuffer;

[0102] iPLEXTerminationmix;

[0103] ExtendPrimerMix;

[0104] iPLEXEnzyme.

[0105] Other reagents

[0106] Resin;

[0107] Chip board;

[0108] Standards (samples containing known genotypes of drug resistance gene loci).

[0109] Experimental procedures

[0110] Sample DNA extraction

[0111] Take 1 mL of sputum sample, add 100 μL of proteinase K (20 mg / mL) and 10 μL of RNase A (10 mg / mL), vortex to mix, and place in a 56°C water bath for 1 hour.

[0112] Add 200 μL of saturated sodium chloride solution, vortex to mix, centrifuge at 12000 rpm for 10 minutes, and take the supernatant.

[0113] Add 200 μL of phenol-chloroform (1:1), vortex to mix, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant.

[0114] Add 500 μL of isopropanol, precipitate at -20°C for 1 hour, centrifuge at 12000 rpm for 10 minutes, and discard the supernatant.

[0115] Add 1 mL of 70% ethanol to wash the precipitate, centrifuge at 12000 rpm for 5 minutes, and discard the supernatant.

[0116] Air-dry the precipitate, add 50 μL TE buffer to dissolve it, and store it at -20°C until use.

[0117] Use a nucleic acid quantifier to measure DNA concentration and purity. The A260 / A280 ratio should be between 1.8-2.0.

[0118] PCR amplification

[0119] Prepare PCR reaction system, each reaction system includes:

[0120] 10xPCRBuffer5μL;

[0121] 25mM MgCl24μL;

[0122] 25mM dNTP Mix 1μL;

[0123] 0.5 μM primer mixture (select corresponding primers according to different gene loci) 5 μL;

[0124] 5U / μL Taq DNA polymerase 1μL;

[0125] DNA template 2 μL;

[0126] Total volume 25 μL.

[0127] The reaction system was placed in a PCR instrument and the reaction program was set: denaturation at 95°C for 2 minutes, followed by 45 cycles, each cycle including denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, and finally extension at 72°C for 5 minutes.

[0128] After amplification, store the reaction tube at 4°C.

[0129] SAP Enzyme Digestion

[0130] Prepare SAP reaction system, each reaction system includes:

[0131] Nanopure Water 1.53 μL;

[0132] SAPBuffer 0.17 μL;

[0133] SAPEnzyme (1.7 U / μL) 0.3 μL;

[0134] 5 μL of PCR amplification product;

[0135] Total volume 7 μL.

[0136] Place the reaction system in a PCR instrument and set the reaction program: digest at 37°C for 40 minutes, inactivate at 85°C for 5 minutes, and then store at 4°C.

[0137] Single base extension reaction

[0138] Prepare the iPLEX extension reaction system. Each reaction system includes:

[0139] Nanopure Water 0.619 μL;

[0140] iPLEX Buffer 0.2 μL;

[0141] iPLEXTerminationmix 0.2 μL;

[0142] ExtendPrimerMix (select corresponding primers according to different gene loci) 0.94 μL;

[0143] iPLEX Enzyme 0.041 μL;

[0144] 7 μL of SAP digestion product;

[0145] Total volume 9 μL.

[0146] The reaction system was placed in a PCR instrument and the reaction program was set: denaturation at 94°C for 30 seconds, followed by 40 cycles, each cycle including denaturation at 94°C for 5 seconds, annealing at 52°C for 5 seconds, extension at 80°C for 5 seconds, and finally extension at 72°C for 3 minutes.

[0147] Product analysis

[0148] Desalting and purification

[0149] The product of the extension reaction was mixed with 15 mg of clean resin and incubated at room temperature for 15 minutes, with gentle inversion during mixing.

[0150] Centrifuge at 3200 g for 5 minutes and collect the supernatant.

[0151] Transfer the supernatant to a new centrifuge tube, add 24 μL Nuclease-free water, and mix gently.

[0152] Mass spectrometry analysis

[0153] The desalted and purified product was transferred to the chip board according to the MassarrayNanodispenser operating instructions.

[0154] Place the chip plate into the Massarray Analyser Four chip slot, with the text-printed side of the chip facing outward, and use tweezers to squeeze the chip toward the lower left.

[0155] Start the mass spectrometer for detection and set the detection parameters according to the instrument manual.

[0156] After the detection is completed, the data is analyzed using the analysis software that comes with the mass spectrometer, and the genotype of the corresponding site is determined based on the peak position corresponding to the product molecular weight.

[0157] Result determination

[0158] Genotype determination

[0159] For each gene locus, the analysis software will display the corresponding mass spectrum peak graph. For example, in the mass spectrum peak graph of the rpoB-533 locus, if a specific peak combination appears, it indicates that the genotype of the locus is the corresponding mutation type.

[0160] Compare the test results with known resistance standards to determine the sample's resistance to the corresponding drug.

[0161] Drug resistance assessment

[0162] Based on the results of genotype determination, the drug resistance of the sample is evaluated. For example, if drug-resistant mutations appear at certain sites of the rpoB gene, it indicates that the strain may be resistant to rifampicin; if drug-resistant mutations appear at certain sites of the katG and inhA genes, it indicates that the strain may be resistant to isoniazid.

[0163] For multidrug-resistant samples, the mutation status of all relevant gene loci was comprehensively analyzed to assess the severity of drug resistance.

[0164] Quality Control

[0165] Positive control: Positive control samples are set in each batch of experiments to ensure the accuracy and reliability of experimental operations. Positive control samples should contain samples with known genotypes of drug-resistant gene loci, and be tested and analyzed at the same time as the samples to be tested.

[0166] Negative control: A negative control sample is set in each batch of experiments, that is, a sample without Mycobacterium tuberculosis, to detect whether there is contamination during the experiment. Negative control samples should be processed and tested at the same time as the samples to be tested to ensure the accuracy of the experimental results.

[0167] Repeatability testing: Repeated testing of some samples should be conducted, and the consistency of the test results should be within an acceptable range to ensure the repeatability and reliability of the experiment.

[0168] Embodiment 2

[0169] A kit for detecting tuberculosis resistance-related genes, including a primer set for tuberculosis resistance-related gene mutations, 25 site information of 7 genes related to tuberculosis resistance, as shown in Table 1, and the primer set as shown in Table 2:

[0170] PCR primers

[0171] According to the 55 gene mutation types of the 7 genes to be detected, amplification primers for each gene mutation type are designed. The amplification primer can amplify a DNA sequence including the mutation site. The amplification primer has 15 or more bases at the 3' end that completely match the target gene sequence, and a universal tag sequence of 10 bases (ACGTTGGATG) at the 5' end. The specific primers and extension primers are shown in Table 2.

[0172] Table 1 Drug resistance gene mutation information

[0173]

[0174]

[0175] Table 2 PCR amplification primers and extension primer sequences

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] Reaction instruments and reagents

[0183] Reaction instrument: MassARRAY nucleic acid mass spectrometry analysis system (MassARRAY Analyzer 4 System, 96 Genotyping (Agena))

[0184] Reagents: Complete Gold Genotyping Reagent Set 10x96 (Agena)

[0185] The detection method is as follows:

[0186] Prepare DNA samples of satisfactory quality

[0187] After sealing and centrifugation, PCR amplification was performed. The PCR amplification reaction system was as shown in Table 3.

[0188] Table 3 PCR amplification reaction system

[0189]

[0190] Note: N / A means no meaning

[0191] The PCR amplification conditions are shown in Table 4:

[0192] Table 4 PCR amplification conditions

[0193]

[0194] SAP enzyme digestion reaction: The PCR amplification product in the previous step was digested using the reaction system in Table 5 to remove excess dNTPs in the reaction. The reaction conditions were: 37°C: 40 minutes, 85°C: 5 minutes, and stored at 4°C for later use.

[0195] Table 5 SAP enzyme digestion reaction conditions

[0196]

[0197] Single nucleotide base extension reaction: After centrifugation of the above reaction product, an extension reaction was performed using the reaction system in Table 6. The reaction conditions are shown in Table 7.

[0198] Table 6 Extension reaction system

[0199]

[0200] Table 7 Extension reaction conditions

[0201]

[0202] Finally, the product is desalted and purified by resin, spotted on the corresponding chip, and the results are automatically analyzed by the analysis software of the mass spectrometer. The genotype of the corresponding site is determined according to the peak position corresponding to the molecular weight of the product.

[0203] Embodiment 3

[0204] The reagents and methods in Example 2 were used to detect 60 clinical samples that had been sequenced for the first generation, and some sites of 6 samples (S234, y11, y54, y81, y126, y252) were selected. See the mass spectrometry peak diagram and the comparison of the first generation sequencing peak diagram (Figure 1-6) for details. It can be seen that the method of the present invention can quickly and accurately detect the genotype of the target site, with strong flexibility and short detection cycle. Clinicians can provide medication guidance based on the test results. Among the 7 genes involved in the present invention, genes related to rifampicin resistance include rpoB, genes related to isoniazid resistance include katG and inhA, genes related to fluoroquinolone resistance include gyrA and gyrB, and genes related to second-line anti-tuberculosis drugs eis and rrs.

[0205] Based on the above interpretation, relevant information on tuberculosis drug resistance can be obtained, thereby improving the rational use of drugs.

[0206] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer set for detecting drug resistance gene loci of Mycobacterium tuberculosis, characterized in that: Includes core gene locus primers and other locus primers; The core gene locus primers include: rpoB gene primers, including but not limited to 533 site (rpoB-533), 511 site (rpoB-511), 513 site (rpoB-513-1), 482 site (rpoB-482), 522 site (rpoB-522), 526 site (rpoB-526-1, rpoB-526-2), 531 site (rpoB-531-1, rpoB-531-2, rpoB-531-3), 516 site (rpoB-516), etc., and corresponding amplification primers and extension primers, the sequences of the extension primers are shown in SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQID NO:71, etc.; inhA gene primers, including amplification primers and extension primers for sites such as (-15) site (inhA-15) and (-8) site (inhA-8); katG gene primers, including amplification primers and extension primers for sites such as site 315 (katG-315); eis gene primers, including amplification primers and extension primers at the (-37) site (eis-promoter-37), (-10) site (eis-promoter-10), (-14) site (eis-promoter-14) and other sites in the promoter region; rrs gene primers, including amplification primers and extension primers for sites 1321 (rrs-1321), 1401 (rrs-1401), and 1300 (rrs-1300); gyrB gene primers, including amplification primers and extension primers for sites 538 (gyrB-538), 500 (gyrB-500), and 540 (gyrB-540); gyrA gene primers, including amplification primers and extension primers at positions 74 (gyrA-74), 90 (gyrA-90), 94 (gyrA-94), and 91 (gyrA-91); The other site primers include other site primers of the rpoB gene, such as primers of sites such as F505V, S512T, and D626D; Primers for other sites of the gyrB gene, such as primers for sites such as N538I, G509C, and T539N; Primers for other sites of the eis gene, such as primers for C(-12)T, C(-2)T, G37T and other sites.

2. A primer set for detecting drug-resistant gene loci of Mycobacterium tuberculosis according to claim 1, characterized in that: The primer set is divided into 3 Wells, as follows: Well1: rpoB-533, gyrA-90, rpoB-511, rpoB-513-1, rpoB-482, rrs-1321, gyrB-538, rrs-14 01. gyrA-74, rrs-1300, rpoB-522, katG-315, rpoB-526-1, gyrA-94, gyrB-500, rpoB-526-2; Well2: rpoB-531-1, rpoB-531-2, rpoB-513-2, rpoB-572, inhA-15, gyrB-540, rpoB-522, gyrA-94; Well3: rpoB-513-3, rpoB-516, eis-promoter-37, eis-promoter-10, eis-promoter-14, rpoB-531-3, rpoB-516, inhA-8, gyrA-91.

3. A primer set for detecting drug-resistant gene loci of Mycobacterium tuberculosis according to claim 2, characterized in that: The primer has 15 or more bases at the 3' end that completely match the target gene sequence, and a universal tag sequence of 10 bases (ACGTTGGATG) at the 5' end.

4. A method for preparing a primer set according to any one of claims 1 to 3, characterized in that: The following steps are involved: Primer design: Based on the latest research results of Mycobacterium tuberculosis resistance genes and known resistance sites, combined with bioinformatics analysis, amplification primers and single-base extension primers for each gene site are designed, taking into account factors such as primer specificity, amplification efficiency, and annealing temperature. Primers are screened and optimized using computer software to ensure that primers can accurately amplify and detect target gene sites. Sequence alignment and homology analysis are performed on the designed primers to avoid cross-hybridization and nonspecific amplification between primers; Primer synthesis: The designed primers are synthesized by chemical synthesis method, and the synthesized primers are subjected to high performance liquid chromatography (HPLC) or other appropriate purification methods to improve the purity and quality of the primers. The concentration and quality of the synthesized primers are measured to ensure that the concentration of the primers meets the requirements of subsequent experiments and are free of impurities and degradation products; Primer ratio: According to the detection requirements and experimental experience of the MassARRAY nucleic acid mass spectrometry platform, the single-base extension primers are accurately ratioed to ensure that the amplification efficiency of each single-base extension primer in the single-hole reaction system is consistent and that the products of different molecular masses after extension can be accurately detected in the flying nucleic acid mass spectrometry; The specific ratio is as follows: Well1: the ratio of rpoB-533 to primer SEQ ID NO:67 was 2.64 μl, the ratio of gyrA-90 to primer SEQ ID NO:68 was 2.89 μl, the ratio of rpoB-511 to primer SEQ ID NO:69 was 3.53 μl, the ratio of rpoB-513-1 to primer SEQ ID NO:70 was 4.41 μl, the ratio of rpoB-482 to primer SEQ ID NO:71 was 4.91 μl, the ratio of rrs-1321 to primer SEQ ID NO:72 was 5.69 μl, the ratio of gyrB-538 to primer SEQ ID NO:73 was 5.96 μl, the ratio of rrs-1401 to primer SEQ ID NO:74 was 6.59 μl, and the ratio of gyrA-74 to primer SEQ ID NO:75 was 7. The ratio of primer SEQ ID NO:75 is 6.95 μl, the ratio of primer SEQ ID NO:76 corresponding to rrs-1300 is 6.99 μl, the ratio of primer SEQ ID NO:77 corresponding to rpoB-522 is 7.01 μl, the ratio of primer SEQ ID NO:78 corresponding to katG-315 is 7.44 μl, the ratio of primer SEQ ID NO:79 corresponding to rpoB-526-1 is 7.62 μl, the ratio of primer SEQ ID NO:80 corresponding to gyrA-94 is 7.71 μl, the ratio of primer SEQ ID NO:81 corresponding to gyrB-500 is 7.97 μl, the ratio of primer SEQ ID NO:82 corresponding to rpoB-526-2 is 8.23 ​​μl, and the water is added to 398.2 μl; Well2: rpoB-531-1 corresponding to primer SEQ ID NO:83 is 2.71 μl, rpoB-531-2 corresponding to primer SEQ ID NO:84 is 3.65 μl, rpoB-513-2 corresponding to primer SEQ ID NO:85 is 4.41 μl, rpoB-572 corresponding to primer SEQ ID NO:86 is 4.52 μl, inhA-15 corresponding to primer SEQ ID NO:87 is 5.89 μl, gyrB-540 corresponding to primer SEQ ID NO:88 is 6.45 μl, rpoB-522 corresponding to primer SEQ ID NO:89 is 5.10 μl, gyrA-94 corresponding to primer SEQ ID NO:90 is 6.37 μl, make up to 461 μl with water; Well3: rpoB-513-3 corresponding to primer SEQ ID NO:91 was 4.41 μl, rpoB-516 corresponding to primer SEQ ID NO:92 was 5.97 μl, eis-promoter-37 corresponding to primer SEQ ID NO:93 was 8.02 μl, eis-promoter-10 corresponding to primer SEQ ID NO:94 was 8.13 μl, eis-promoter-14 corresponding to primer SEQ ID NO:95 was 9.48 μl, rpoB-531-3 corresponding to primer SEQ ID NO:96 was 3.51 μl, rpoB-516 corresponding to primer SEQ ID NO:97 was 5.32 μl, inhA-8 corresponding to primer SEQ ID NO:98 was 6.28 μl, gyrA-91 corresponding to primer SEQ ID NO:99 was 7.84 μl, and gyrA-10 corresponding to primer SEQ ID NO:91 was 6.20 μl. The ratio of NO:99 is 6.63μl, add water to 442μl.

5. A method for detecting drug resistance gene loci of Mycobacterium tuberculosis using the primer set according to any one of claims 1 to 3, characterized in that: The following steps are involved: Sample preparation: Collect clinical samples, such as sputum, blood, urine, etc. The samples should be as fresh as possible to avoid contamination and degradation. Then use conventional DNA extraction methods, such as phenol-chloroform method, kit extraction method, etc., to extract the DNA of Mycobacterium tuberculosis from the samples. Determine the concentration and purity of the extracted DNA to ensure that the DNA quality meets the detection requirements, such as the DNA concentration should be greater than 50ng / μl, and the A260 / A280 ratio should be between 1.8-2.0; PCR amplification: Prepare the PCR reaction system, mix the extracted DNA, primers, PCR enzyme, buffer, dNTP, etc. in a certain proportion to ensure the accuracy and stability of the reaction system, and perform PCR amplification reaction. The reaction conditions are: denaturation at 95°C for 2 minutes, followed by 45 cycles, each cycle including denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, and finally extension at 72°C for 5 minutes. During the amplification process, attention should be paid to controlling the reaction temperature and time to avoid abnormalities in the PCR reaction; SAP enzyme digestion: SAP enzyme digestion is performed on the PCR amplification product to remove excess dNTPs in the reaction and reduce interference with the extension reaction. The SAP reaction system is prepared by mixing the PCR amplification product, SAP enzyme, buffer, etc. in a certain proportion and performing SAP enzyme digestion reaction. The reaction conditions are: digestion at 37°C for 40 minutes, inactivation at 85°C for 5 minutes, and then storage at 4°C. After the digestion reaction is completed, the subsequent extension reaction should be carried out in time to avoid DNA degradation; Single base extension reaction: Use single base extension primer to perform single base extension reaction on the digestion product to obtain extension product, prepare iPLEX extension reaction system, mix the SAP digestion product, iPLEX enzyme, extension primer, buffer, etc. in a certain proportion, and perform single base extension reaction. The reaction conditions are: denaturation at 94℃ for 30 seconds, followed by 40 cycles, each cycle includes denaturation at 94℃ for 5 seconds, annealing at 52℃ for 5 seconds, extension at 80℃ for 5 seconds, and finally extension at 72℃ for 3 minutes. During the extension reaction, the reaction temperature and time should be strictly controlled to ensure the accuracy of the extension reaction. Product analysis: Desalting and purifying the extension products to remove residual salt ions in the reaction and improve the accuracy of mass spectrometry analysis. Using nucleic acid mass spectrometry analysis software to type the sites and determine whether there are mutations. Compare the test results of the samples with known drug resistance standards to analyze the type and degree of drug resistance of the samples. Record and analyze the test results and generate a test report to provide a reference for clinical treatment.

6. The detection method according to claim 5, characterized in that: The volume ratio of the PCR amplification reaction solution, the SAP digestion reaction solution and the iPLEX reaction solution was 5:2:

2.

7. Use of the primer set according to any one of claims 1 to 3 in preparing a kit for detecting drug resistance of Mycobacterium tuberculosis.

8. A Mycobacterium tuberculosis drug resistance detection kit, characterized in that: The kit comprises the primer set according to any one of claims 1 to 3 and a single base extension primer prepared according to the primer ratio scheme according to claim 4; the kit comprises: Primer reagents: Contains various amplification primers and single-base extension primers, which are packed in different tubes and marked with primer names and sequence information; PCR reaction reagents: including 10xPCRBuffer, 25mMMgCl2, 25mMdNTPMix, PCREnzyme, etc., prepared in a certain proportion and packaged in the kit; SAP enzyme reaction reagents: including NanopureWater, SAPBuffer, SAPEnzyme, etc., prepared in a certain proportion and packaged in the kit; iPLEX reaction reagents: including iPLEX Buffer, iPLEX Termination Mix, Extend Primer Mix, iPLEX Enzyme, etc., prepared in a certain proportion and packaged in the kit; Other reagents: such as resin, chip boards, standards, etc., are prepared according to experimental needs. Method for using the kit Sample processing: Collect and process samples according to the instructions to extract DNA of Mycobacterium tuberculosis; PCR amplification: Mix the extracted DNA, primer reagents, and PCR reaction reagents in a certain proportion to perform PCR amplification reaction; SAP enzyme digestion: The PCR amplification product was digested with SAP enzyme; Single base extension reaction: Use single base extension primer to perform single base extension reaction on the digestion product; Product analysis: The extension products were desalted and purified, and then detected and analyzed using nucleic acid mass spectrometry software.

9. Use of the primer set or kit according to any one of claims 1 to 8 in detecting drug resistance of Mycobacterium tuberculosis, characterized in that: The applications include but are not limited to the following: Clinical diagnosis: used to detect drug resistance of Mycobacterium tuberculosis in clinical samples to provide a basis for clinical treatment; Drug research and development: assist drug research and development companies in drug screening and drug resistance mechanism research, and develop new anti-tuberculosis drugs; Public health monitoring: used for monitoring, prevention and control of tuberculosis, understanding the drug resistance of Mycobacterium tuberculosis, and formulating reasonable prevention and control strategies.

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