Primer group for detecting mycobacterium tuberculosis complex and drug resistance thereof and application of primer group

Abstract

The invention relates to the field of nucleic acid detection, and provides a primer group for detecting a mycobacterium tuberculosis complex group and drug resistance thereof and application of the primer group. According to the method for detecting the drug resistance of the mycobacterium tuberculosis complex as well as kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin and moxifloxacin) and linezolid, provided by the invention, specific detection on the mutation sites and specific sequences can be realized; the method has the advantages of high accuracy, high sensitivity, good repeatability, low cost, short detection period, visual result, no need of signal intervention and the like. The method is more in line with the gene mutation characteristics of the mycobacterium tuberculosis drug-resistant strain, overcomes the defect that the number of SNP sites detected at a time in the prior art is small, has the advantages of being high in accuracy and sensitivity, good in repeatability, low in cost and short in detection period, and has a very good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid detection, in particular to a primer set for detecting Mycobacterium tuberculosis complex and its drug resistance and application. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium tuberculosis complex. It is the infectious disease with the highest number of deaths from a single infectious source. my country is one of the 30 countries with a high burden of drug-resistant tuberculosis. Timely, rapid and accurate detection of drug sensitivity of Mycobacterium tuberculosis complex can reduce the occurrence, spread and adverse prognosis of drug-resistant tuberculosis. Second-line anti-tuberculosis drugs refer to anti-tuberculosis drugs other than first-line drugs such as isoniazid, rifampicin, ethambutol and pyrazinamide. At present, there are many methods for detecting the sensitivity of first-line anti-tuberculosis drugs, while there are fewer methods for detecting the sensitivity of second-line anti-tuberculosis drugs. The types of second-line anti-tuberculosis drugs are limited, the cost is relatively high, and the side effects are large. Patients with first-line drug-resistant tuberculosis can test the sensitivity of strains to second-line drugs before choosing drugs, which can reduce the occurrence of new drug resistance, avoid unnecessary expenses and damage to the body, and guide the selection of appropriate drug combinations to increase the probability of successful treatment.

[0003] Currently, commonly used methods for drug resistance detection include phenotypic drug sensitivity methods and molecular detection methods. Phenotypic drug sensitivity methods include the traditional ratio method and absolute concentration method based on Roche culture medium (about 28 days), the BACTECMGIT960 rapid drug sensitivity culture method based on liquid culture medium (about 14 days), and the microplate microbroth dilution method (7-14 days) that has emerged in recent years. Such phenotypic detection methods require the pre-obtaining of pure cultures of Mycobacterium tuberculosis complex, which is time-consuming and cumbersome to detect. Some methods require expensive reagents and equipment. Currently, commonly used molecular detection methods include real-time fluorescence quantitative PCR (melting curve method, probe method), biochip method, linear probe method and dot hybridization method. Some of these methods can directly detect clinical samples, but most methods can only detect Mycobacterium tuberculosis complex cultures. The detection time is short, about 3h~3 days, but most methods have insufficient detection throughput and limited drug resistance. The second-generation sequencing technology and the third-generation long-read sequencing technology (including single-molecule real-time sequencing platform and Oxford nanopore sequencing technology platform) have the advantages of high sensitivity and reusable sequencing data analysis in detecting tuberculosis drug resistance. However, they are limited by factors such as high cost and difficult operation, and are not promoted in some areas with limited resources. Summary of the invention

[0004] The purpose of the present invention is to provide a primer set for detecting Mycobacterium tuberculosis complex and its drug resistance and application.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a primer set for detecting Mycobacterium tuberculosis complex and its drug resistance, including a primer set for detecting Mycobacterium tuberculosis complex IS6110 The primer pair of the gene, the nucleotide sequence is SEQ ID NO: 1-2; used to detect kanamycin, amikacin, capreomycin resistance genes rrs The primer pair has a nucleotide sequence of SEQ ID NO: 3-4; used to detect kanamycin and amikacin resistance gene fragments eis Primer pair for promoter, nucleotide sequence is SEQ ID NO:5-6; used for detecting fluoroquinolone (ofloxacin, levofloxacin, moxifloxacin) resistance gene yrA The primer pair has a nucleotide sequence of SEQ ID NO: 7-8; used to detect linezolid resistance gene R The primer pair has a nucleotide sequence of SEQ ID NO: 9-10.

[0006] In a second aspect, the present invention provides a primer combination for detecting Mycobacterium tuberculosis complex and its drug resistance based on MassArray nucleic acid mass spectrometry, comprising the primer set and a single base extension primer set for detecting the following 13 gene loci: IS6110-1014 , rrs1401 , rrs1402 , rrs1484 , eis-10, eis-12, eis-14, gyrA94_AC, gyrA94G_C, gyrA90G_G, gyrA91_CG, rplC154_GT and internal control IC-157 , and their nucleotide sequences are shown in SEQ ID NO: 13-25, respectively.

[0007] In a third aspect, the present invention provides a detection reagent or a kit containing the primer set or the primer combination.

[0008] In a fourth aspect, the present invention provides a kit for detecting Mycobacterium tuberculosis complex and its drug resistance based on MassArray nucleic acid mass spectrometry, the kit comprising the primer combination, dNTPs, Mg 2+ , DNA polymerase, shrimp alkaline phosphatase, resin purification solution and reaction buffer, etc.

[0009] In a fifth aspect, the present invention provides the use of the primer set, the primer combination or the detection reagent or kit in detecting Mycobacterium tuberculosis complex and its drug resistance (including non-disease diagnosis and treatment purposes).

[0010] In a sixth aspect, the present invention provides use of the primer set or the primer combination in preparing a detection reagent or a kit for detecting Mycobacterium tuberculosis complex and its drug resistance.

[0011] In a seventh aspect, the present invention provides a method for detecting Mycobacterium tuberculosis complex and its drug resistance based on MassArray nucleic acid mass spectrometry (including non-disease diagnosis and treatment purposes), comprising the following steps: (1) using the sample DNA to be tested as a template and using the primer set to perform a multiplex PCR amplification reaction to obtain an amplified product; (2) digesting the amplified product of step (1) with shrimp alkaline phosphatase (SAP) to obtain a digestion product; (3) performing a single base extension reaction on the digestion product of step (2) using the single base extension primer to obtain an extension product; (4) desalting and purifying the extension product of step (3) to obtain a purified product; (5) Analyze the purified product of step (4) using the MassArray nucleic acid mass spectrometry analysis system.

[0012] Preferably, in the reaction system used in step (1), the concentration of each primer is 0.6-1.3 μM, and preferably the concentration of each primer is 1 μM.

[0013] Preferably, the reaction procedure used in step (1) is: 95°C for 2 min; 95°C for 10 s, 60-66°C for 30 s, 72°C for 30 s, 40 cycles; 72°C for 5 min; more preferably, the reaction procedure is: 95°C for 2 min; 95°C for 10 s, 62°C for 30 s, 72°C for 30 s, 40 cycles; 72°C for 5 min.

[0014] Preferably, in the reaction system used in step (3), the concentration of the primers shown in SEQ ID NOs: 13-25 is 7-18 μM, and preferably the concentrations of the primers shown in SEQ ID NOs: 13-25 are 11.5 μM, 13 μM, 7.0 μM, 13 μM, 10.5 μM, 15 μM, 6 μM, 13.5 μM, 18 μM, 10 μM, 14 μM, 12 μM, and 15 μM, respectively.

[0015] Preferably, the reaction procedure used in step (3) is: 94°C for 3 min; 94°C for 15 s, 50-55°C for 15 s, 72°C for 10 s, 50 cycles; 72°C for 5 min; Preferably, the reaction procedure is: 94°C for 3 min; 94°C for 15 s, 52°C for 15 s, 72°C for 10 s, 50 cycles; 72°C for 5 min.

[0016] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) The method provided by the present invention for detecting Mycobacterium tuberculosis complex and its resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid can achieve specific detection of the above mutation sites and specific sequences, and has the advantages of high accuracy, high sensitivity, good repeatability, low cost, short detection cycle, intuitive results and no need for bioinformatics intervention.

[0017] (ii) The present invention overcomes the defect of the prior art that only a small number of SNP (single nucleotide polymorphism) sites can be detected at one time, and has low cost and a short detection cycle, and is suitable for wide promotion; the samples required by the present invention have strong adaptability, and can detect both Mycobacterium tuberculosis complex cultures and suspected clinical sputum samples of tuberculosis well.

[0018] (III) The present invention is applicable to suspected and confirmed tuberculosis patients, and can help diagnose tuberculosis and predict the patient's resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid, guide clinical medication, improve treatment effects, and block the spread of drug-resistant tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is an agarose gel electrophoresis diagram of multiple PCR products (extension temperature test) in a preferred embodiment of the present invention.

[0020] Figure 2 The figure is an agarose gel electrophoresis diagram of multiplex PCR products (primer concentration test) in a preferred embodiment of the present invention.

[0021] Figure 3 This is an agarose gel electrophoresis diagram of multiple PCR products (tested in different systems) in a preferred embodiment of the present invention.

[0022] Figure 4 This is a mass spectrum diagram of the test of primer concentration in a single base extension reaction in a preferred embodiment of the present invention.

[0023] Figure 5 This is a mass spectrum diagram of the test of primer concentration in a single base extension reaction in a preferred embodiment of the present invention.

[0024] Figure 6 This is a test mass spectrum of primer replacement in a single base extension reaction in a preferred embodiment of the present invention.

[0025] Figure 7 This is a test mass spectrum of primer replacement in a single base extension reaction in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0026] The present invention aims to provide a molecular detection method for Mycobacterium tuberculosis complex and its resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid, which can detect the gene mutation combination related to the resistance of Mycobacterium tuberculosis complex to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid more comprehensively, and can significantly improve the sensitivity of resistance detection. The present invention adopts the following technical scheme: The present invention provides a primer set for detecting Mycobacterium tuberculosis complex and its resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid based on MassArray nucleic acid mass spectrometry. The primer set sequence includes multiple amplification primers (sequences are shown in SEQ ID NOs: 1-12, respectively) and extension primers (sequences are shown in SEQ ID NOs: 13-25).

[0027] The present invention also provides a method for detecting Mycobacterium tuberculosis complex and its resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid based on MassArray nucleic acid mass spectrometry. The method comprises using a primer set for detecting Mycobacterium tuberculosis complex and its resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid to perform PCR amplification and single base extension reaction on the genome of a suspected Mycobacterium tuberculosis isolate or a suspected tuberculosis clinical sample to be tested, and then using the MassArray nucleic acid mass spectrometry platform to detect the reaction product to determine the genotype of the SNP site in the genome of the object to be tested.

[0028] The present invention also provides the use of the primer set and the primer combination in preparing a product for detecting the Mycobacterium tuberculosis complex and its resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid, including a composition / reagent / kit for detecting the Mycobacterium tuberculosis complex and its resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid.

[0029] The primer sets and primer combinations for SNP sites for detecting resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid of Mycobacterium tuberculosis complex provided by the present invention are obtained through a large number of clinical sample tests and screening, and combined with the recommendations of the "List of Gene Mutations Related to Drug Resistance of Mycobacterium Tuberculosis Complex" issued by the World Health Organization, can achieve accurate typing of point mutations and small fragment deletion / insertion mutation samples; The SNP site primer set provided by the present invention covers 11 sites of four common genes related to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid resistance of Mycobacterium tuberculosis complex, including rrs (Kanamycin, Amikacin, Capreomycin resistance): rrs1401 , rrs1402 , rrs1484;eis Promoter (Kanamycin and Amikacin Resistance): eis-10, eis-12, eis-14; gyrA [Fluoroquinolone (ofloxacin, levofloxacin, moxifloxacin) resistance]: gyrA94_AC, gyrA94G_C, gyrA90G_G, gyrA91_ CG; rplC (Linezolid resistance): rplC154_GT . It is based on the characteristics of drug-resistant mutations of clinical Mycobacterium tuberculosis in my country and the point mutation sites recommended and selected according to the "List of Gene Mutations Associated with Drug Resistance in Mycobacterium Tuberculosis Complex" published by the World Health Organization in 2022 and 2023. The site selection is more accurate.

[0030] The SNP site primer set provided by the present invention covers specific sequences for identifying the Mycobacterium tuberculosis complex. One site in the specific sequence is assumed to be a SNP site for detection, and the Mycobacterium tuberculosis complex can be detected by sequence molecular weight.

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0032] Example 1 Design of primer sets for detecting Mycobacterium tuberculosis complex and its drug resistance The present invention provides a primer set for IS6110 for Mycobacterium tuberculosis complex and rrs (kanamycin, amikacin, capreomycin resistance) for seven drug resistance detections, eis promoter (kanamycin and amikacin), gyrA [fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin)] and rplC (linezolid) and internal control (IC), a total of five genes; and also includes 13 sites, namely rrs1401, rrs1402, rrs1484; eis-10, eis-12, eis-14; gyrA94_AC, gyrA94G_C, gyrA90G_G, gyrA91_CG; rplC154_GT; IS6110-1014; IC-157; in this embodiment, the sequences of the amplification primer set are shown in SEQ ID NOs: 1-12, respectively (Table 1).

[0033] Table 1 Primer sequences for SNP site amplification

[0034] When an extension primer is required in actual application, the primer set further includes 13 single-base extension primers shown in SEQ ID NOs: 13-25 (Table 2).

[0035] Table 2 Sequences of single base extension primers at SNP sites

[0036] Example 2 Method for detecting Mycobacterium tuberculosis complex and its kanamycin, amikacin, capreomycin, fluoroquinolone (ofloxacin, levofloxacin, moxifloxacin) and linezolid resistance SNP sites This embodiment provides a method for detecting Mycobacterium tuberculosis complex and its kanamycin, amikacin, capreomycin, fluoroquinolone (ofloxacin, levofloxacin, moxifloxacin) and linezolid resistance SNP sites, the method comprising using the amplification primer set and the single base extension primer set in Example 1 to detect the nucleotides at the SNP site in the genome of the sample to be tested.

[0037] The primer set in Example 1 can be used to detect 12 sites of 4 common genes related to the identification of Mycobacterium tuberculosis complex, kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid resistance, including point mutations and small fragment deletion mutation sites, with the characteristics of high accuracy, high sensitivity, good repeatability, low cost, short detection cycle, intuitive results and no need for bioinformatics intervention.

[0038] Specifically, the above primer set is used to perform PCR amplification and base extension reaction on the genome of the sample to be tested, and then the MassARRAY system is used to detect the product obtained by the reaction to determine the nucleotide of the SNP site in the genome of the sample to be tested.

[0039] The main reagent information used in the examples is as follows: The main instrument information used in the examples is as follows: Detection principle: 1) Selection of resistance detection sites for Mycobacterium tuberculosis complex: Based on the sites related to the detection of kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid resistance in Mycobacterium tuberculosis complex that have been disclosed in the prior art, the "List of Gene Mutations Associated with Resistance of Mycobacterium tuberculosis Complex" published by the World Health Organization in 2022 and 2023, and point mutation sites selected based on the characteristics of clinical Mycobacterium tuberculosis resistance mutations in my country, a combination of resistance detection sites with high sensitivity is selected.

[0040] 2) Selection of genes and sites for detection of Mycobacterium tuberculosis complex: According to the characteristics of Mycobacterium tuberculosis complex, a specific sequence in the Mycobacterium tuberculosis complex-specific gene IS6110 was selected, and the 1041st site of the specific sequence was assumed to be a SNP site for detection, and the Mycobacterium tuberculosis complex was detected by sequence molecular weight. The specific genes and sites are shown in Table 3.

[0041] Table 3 Genotypes of Mycobacterium tuberculosis complex and its resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid

[0042] Primer design and establishment of a preliminary reaction system: First, the present invention uses oligo software to adjust relevant parameters to complete the preliminary design of multiple PCR primers for 5 genes and extension primers for 13 sites, manually calculate the molecular weight of the extension primers and adjust the difference between each extension primer in a single reaction to >40, while ensuring that there is no mutual influence between the extension primers in a single reaction. Synthesize multiple PCR primers, dilute to 100 μM, and configure amplification primers MIX according to the table so that the concentration of each primer in MIX is as shown in Table 4.

[0043] Table 4 Concentrations of primer mixes for multiplex PCR for detection of Mycobacterium tuberculosis complex and its resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid

[0044] Synthesize extension primers, configure extension primer MIX according to Table 5 and adjust it to meet the detection requirements.

[0045] Table 5 Concentration and molecular weight of the extended primer MIX for detection of Mycobacterium tuberculosis complex and its resistance to kanamycin, amikacin, capreomycin, fluoroquinolones (ofloxacin, levofloxacin, moxifloxacin) and linezolid

[0046] The specific steps for primer testing and optimization are as follows: 1) Multiplex PCR: Dilute the sample DNA to 10-15 ng / μL, and prepare the reaction system according to Table 6 and label it, cover the lid, centrifuge, and perform PCR thermal cycling (Table 7). Figure 1 Agarose gel electrophoresis of multiple PCR products.

[0047] Table 6 Multiplex PCR amplification reaction system configuration table Table 7 PCR reaction program

[0048] 2) Shrimp alkaline phosphatase digestion (SAP): Quickly separate the PCR tube, carefully open the lid, add the prepared SAP reaction solution (Table 8) to the reaction well, 2 μL per well; cover the lid, centrifuge, and perform SAP reaction (Table 9). See Tables 8 and 9 for the system and thermal cycle program.

[0049] Table 8 SAP reaction system Table 9 SAP reaction procedure

[0050] 3) Single base extension reaction: Instantly separate the PCR tube, open the lid, add the prepared extension reaction solution (Table 10) to the corresponding reaction well, 3μL per well; cover the lid, centrifuge, and perform single base extension reaction (Table 11). See Tables 10 and 11 for the system and thermal cycle program.

[0051] Table 10 Single base extension reaction system

[0052] Table 11 Single base reaction program

[0053] 4) Sample desalting: Oscillate and mix the resin purification solution, and add 18μL to each reaction; the resin is easy to settle, so oscillate and mix once every 5 seconds. Place the sample in a flip mixer and mix at 20rpm for 30min. After mixing, centrifuge at 2000rpm for 1min, and take the supernatant for testing. 5) Spot the sample on the chip: Add 0.5μL of matrix in the middle of the matching target plate well, wait for at least 20min, and after it is fully dried, add 0.7μL of supernatant, wait for at least 20min, fully dry and crystallize, and measure on the machine. 6) Use MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometer to obtain data.

[0054] Example 3 Optimization of multiplex PCR reaction conditions 1. Test of multiple PCR annealing temperature: dilute the sample DNA to 10-15 ng / μL, configure the reaction system and label it according to Table 6, cover the lid, centrifuge, and perform PCR thermal cycling (Table 12). In this step, set four temperatures of 60, 62, 64, and 66°C. Figure 1 This is the agarose gel electrophoresis diagram of multiple PCR products (extension temperature test). It can be seen from the figure that amplification can be successful at 58~64℃, and the amplification efficiency is highest at 62℃.

[0055] Table 12 Multiplex PCR reaction program (annealing temperature test)

[0056] 2. Test of multiple PCR primer concentration: dilute the sample DNA to 10-15 ng / μL, configure the reaction system and label according to Table 13, involving 4 primer concentrations in total, add 0.4, 0.8, 1.2 and 1.6 μL of PCR primer Mix respectively (the corresponding primer final concentrations are 0.5, 1, 1.5 and 2 μΜ respectively), cover the lid, centrifuge, and perform PCR thermal cycling (Table 7). Figure 2 It is an agarose gel electrophoresis diagram of multiple PCR products (primer concentration test). It can be seen from the figure that the amplification effects are good at 0.5μM, 1μM, and 1.5μM test concentrations. The concentration used in the present invention is 1μM.

[0057] Table 13 Multiplex PCR reaction program (primer concentration test) 3. Test of different multiplex PCR systems: Dilute the sample DNA to 10-15 ng / μL, prepare 5μL and 25μL reaction systems according to Table 6 and Table 14 respectively and label them, cover the lid, centrifuge, and perform PCR thermal cycling (Table 7). Figure 3 It is an agarose gel electrophoresis diagram of multiple PCR products (tested in different systems). It can be seen from the figure that the 5μL reaction system is better. The present invention adopts a 5μL reaction system.

[0058] Table 14 Multiplex PCR reaction procedures (tested in different systems) Example 4 Optimization of single base extension primers 1. Test of single base extension primer concentration: Multiplex PCR, shrimp alkaline phosphatase digestion (SAP) unchanged. Single base extension primer concentration change: The concentration ratio of the primers shown in SEQ ID NO: 13-25 is 11.5μM, 13μM, 7.0μM, 13μM, 10.5μM, 15μM, 6μM, 13.5μM, 18μM, 10μM, 14μM, 12μM, 15μM, and the single base extension is as follows Figure 4 The concentration ratio of the primers shown in SEQ ID NO: 13-25 is 12.5 μM, 13 μM, 7.0 μM, 13 μM, 10.5 μM, 15 μM, 4 μM, 13.5 μM, 17 μM, 10 μM, 13 μM, 12 μM, and 19 μM, respectively. Figure 5 . Ratio one peak intensity is more appropriate.

[0059] 2. Single base extension primer replacement test: multiplex PCR, shrimp alkaline phosphorylase digestion (SAP) unchanged. Single base extension primer SEQ ID NO: 13-25, respectively rrs1401, rrs1402, rrs1484; eis-10, eis-12, eis- 14. gyrA94_AC, gyrA94G_C, gyrA90G_G, gyrA91_CG, rplC154_GT, IS6110-1014, IC-157, IS6110-1041, Extension primer size ， The size of the product after extension is shown in Table 5. The peak diagram after single base extension is shown in Figure 6 。 IC-157 replaced by IC-157R , IS6110-1041 Replace with IS6110-1041F , the peak diagram after single base extension is as follows Figure 7 After replacement, the overall extension peak is not uniform, so it is preferred not to perform replacement.

[0060] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A primer set for detecting Mycobacterium tuberculosis complex and its drug resistance, characterized in that: Includes detection of Mycobacterium tuberculosis complex IS6110 Gene primer pair, nucleotide sequence is SEQ ID NO: 1-2; used to detect kanamycin, amikacin, capreomycin resistance genes rrs The primer pair has a nucleotide sequence of SEQ ID NO: 3-4; used to detect kanamycin and amikacin resistance gene fragments eis The primer pair for detecting the promoter, the nucleotide sequence is SEQ ID NO: 5-6; the primer pair for detecting the fluoroquinolone (ofloxacin, levofloxacin, moxifloxacin) resistance gene gyrA, the nucleotide sequence is SEQ ID NO: 7-8; the primer pair for detecting the linezolid resistance gene R The primer pair for detecting the internal control IC has a nucleotide sequence of SEQ ID NOs: 9-10; the primer pair for detecting the internal control IC has a nucleotide sequence of SEQ ID NOs: 11-12.

2. A primer combination for detecting Mycobacterium tuberculosis complex and its drug resistance based on MassArray nucleic acid mass spectrometry, characterized in that: The invention comprises the primer set of claim 1 and single base extension primers for detecting the following 13 gene loci: IS6110-1014, rrs1401, rrs1402, rrs1484, eis-10, eis-12, eis-14, gyrA94_AC, gyrA94G_C, gyrA90G_G, gyrA91_CG, rplC154_GT Internal control IC-157 , whose nucleotide sequences are shown in SEQ ID NO: 13-25 respectively 。 3. A detection reagent or kit comprising the primer set according to claim 1 or the primer combination according to claim 2.

4. A kit for detecting Mycobacterium tuberculosis complex and its drug resistance based on MassArray nucleic acid mass spectrometry, characterized in that: The kit comprises the primer combination according to claim 2, dNTPs, Mg 2+ , DNA polymerase, shrimp alkaline phosphatase, resin purification solution and reaction buffer.

5. Use of the primer set according to claim 1, the primer combination according to claim 2, or the detection reagent or kit according to claim 3 in detecting Mycobacterium tuberculosis complex and its drug resistance; The applications are for non-disease diagnosis and treatment purposes.

6. Use of the primer set according to claim 1 or the primer combination according to claim 2 in the preparation of a detection reagent or kit for detecting Mycobacterium tuberculosis complex and its drug resistance.

7. A method for detecting Mycobacterium tuberculosis complex and its drug resistance based on MassArray nucleic acid mass spectrometry, characterized in that: The following steps are involved: (1) Using the sample DNA to be tested as a template, performing a multiplex PCR amplification reaction using the primer set described in claim 1 to obtain an amplified product; (2) digesting the amplified product of step (1) with shrimp alkaline phosphatase (SAP) to obtain a digestion product; (3) performing a single base extension reaction on the digestion product of step (2) using the single base extension primer described in claim 2 to obtain an extension product; (4) desalting and purifying the extension product of step (3) to obtain a purified product; (5) analyzing the purified product of step (4) using a MassArray nucleic acid mass spectrometry analysis system; The method is for non-disease diagnosis and treatment purposes.

8. The method according to claim 7, characterized in that In the reaction system used in step (1), the concentration of each primer is 0.6-1.3 μM, preferably the concentration of each primer is 1 μM; In the reaction system used in step (3), the concentration of the primers shown in SEQ ID NOs: 13-25 is 7 to 18 μM, and preferably the concentrations of the primers shown in SEQ ID NOs: 13-25 are 11.5 μM, 13 μM, 7.0 μM, 13 μM, 10.5 μM, 15 μM, 0.6 μM, 13.5 μM, 18 μM, 1 μM, 14 μM, 12 μM, and 15 μM, respectively.

9. The method according to claim 7, characterized in that: The reaction program used in step (1) was: 95°C for 2 min; 95°C for 10 s, 60-66°C for 30 s, 72°C for 30 s, 40 cycles; 72°C for 5 min; Preferably, the reaction procedure is: 95°C for 2 min; 95°C for 10 s, 62°C for 30 s, 72°C for 30 s, 40 cycles; 72°C for 5 min.

10. The method according to claim 7, characterized in that The reaction program used in step (3) was: 94°C for 3 min; 94°C for 15 s, 50-55°C for 15 s, 72°C for 10 s, 50 cycles; 72°C for 5 min; Preferably, the reaction procedure is: 94°C for 3 min; 94°C for 15 s, 52°C for 15 s, 72°C for 10 s, 50 cycles; 72°C for 5 min.

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