Primer composition and application thereof

Abstract

The invention discloses a primer composition and application thereof. According to the invention, a group of primer compositions is obtained aiming at mycobacterium tuberculosis complex group identification and drug resistance related genes thereof. The primer composition can be combined with nucleic acid matrix-assisted laser desorption ionization time-of-flight mass spectrometry to detect a mycobacterium tuberculosis complex group, and can also detect the mutation conditions of 18 drug-resistant genes and 92 gene mutations related to drug resistance of 21 common first-line and second-line antituberculous drugs / types in the mycobacterium tuberculosis complex group. The medication is guided. Based on a MassARRAY technical platform, the primer composition has the advantages of good accuracy, high sensitivity, short required time and high efficiency when being used for detecting the mycobacterium tuberculosis complex group and the drug resistance thereof, is suitable for various sample types, can meet clinical detection requirements, is beneficial to clinical diagnosis and medication guidance of mycobacterium tuberculosis, and realizes precise treatment of pulmonary tuberculosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular detection, and more specifically, relates to a primer composition and application thereof. Background Art

[0002] Tuberculosis (TB) is caused by Mycobacterium tuberculosis complex ( Mycobacterium Tuberculosis Tuberculosis is a chronic respiratory infectious disease caused by Mycobacterium tuberculosis Complex (MTBC) infection. It is also the infectious disease with the highest single mortality rate. All organs in the body can be affected, with lung infection being the most common, namely "pulmonary tuberculosis". Tuberculosis is one of the important public health issues that threaten human health. The key to reducing the incidence and mortality of tuberculosis is to use detection methods to achieve early detection and early treatment of tuberculosis. With the increasing incidence of drug-resistant and multi-drug-resistant tuberculosis, the development of detection products that can accurately detect the drug resistance of Mycobacterium tuberculosis complex is of great significance in the treatment of tuberculosis.

[0003] At present, the products for detecting drug-resistant gene mutations of Mycobacterium tuberculosis on the market in China are mainly for rifampicin and isoniazid resistance. There are few drug-resistant detection products for other anti-tuberculosis drugs, such as cycloserine (Cs), bedaquilin (Bdq) and clofazinmine (Cfz). In addition, the applicable sample types of existing Mycobacterium tuberculosis drug-resistant gene mutation detection products are single, about 80% of which are culture samples, which have the disadvantages of complex operation and greatly increased waiting time for patients. Due to the diverse types of drug-resistant tuberculosis and the complex mutation of related drug-resistant genes, the current products can no longer meet clinical needs well, and there may be risks such as non-detection. Taking into account the current status of tuberculosis epidemic, market demand and the necessity of clinical diagnosis and rational treatment of drug-resistant tuberculosis, it is urgent to establish sensitive, rapid, efficient, flexible, stable, high-throughput, low-cost, and applicable to multiple sample types of Mycobacterium tuberculosis complex detection and drug-resistant tuberculosis detection products and methods, which are the key to clinical rational drug use and control of disease epidemics.

[0004] Nucleic acid matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a molecular detection technology that has developed rapidly in recent years and has been widely used in the fields of tumor resistance gene mutation detection and pathogen typing detection. The MassARRAY DNA flight mass spectrometry analysis system platform (Agena Bioscience) is a nucleic acid mass spectrometry high-precision DNA qualitative and quantitative analysis platform. By combining nucleic acid mass spectrometry technology with multiple PCR amplification technology, it can quickly analyze hundreds of gene mutations at the same time. Although the detection of Mycobacterium tuberculosis complex and drug-resistant tuberculosis based on the MassARRAY DNA flight mass spectrometry analysis system platform has the advantages of high throughput and stable performance, the acquisition of multiple PCR primers will become very difficult as the number of drug-resistant genes and mutations to be detected increases. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a primer combination, which can be used in combination with nucleic acid matrix-assisted laser desorption ionization time-of-flight mass spectrometry to detect the Mycobacterium tuberculosis complex, and can also detect the mutation status of 18 drug-resistant genes and 92 gene mutations related to resistance to 21 types of commonly used first-line and second-line anti-tuberculosis drugs in the Mycobacterium tuberculosis complex.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention aims at identifying the target gene IS6110 of the Mycobacterium tuberculosis complex group and the genes and mutation sites related to the resistance of the Mycobacterium tuberculosis complex group to 21 kinds / types of commonly used first-line and second-line anti-tuberculosis drugs, and obtains a set of primer compositions that can be combined with nucleic acid matrix-assisted laser desorption ionization time-of-flight mass spectrometry to detect the Mycobacterium tuberculosis complex group, and can also detect the mutation of 18 drug-resistant genes and 92 gene mutations related to the resistance of 21 kinds / types of commonly used first-line and second-line anti-tuberculosis drugs in the Mycobacterium tuberculosis complex group. Therefore, the present invention requests protection for the primer composition and its application.

[0007] The present invention provides a primer composition, comprising multiplex PCR amplification primer sets 1 to 4 and single base extension primer sets 1 to 4; The multiplex PCR amplification primer set 1 contains 16 amplification primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 1 to 2 and SEQ ID NOs. 7 to 20, respectively; the multiplex PCR amplification primer set 2 contains 14 amplification primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 1 to 2, SEQ ID NOs. 7 to 10, and SEQ ID NOs. 13 to 20, respectively; the multiplex PCR amplification primer set 3 contains 26 amplification primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 1 to 2, SEQ ID NOs. 7 to 10, SEQ ID NOs. 13 to 18, SEQ ID NOs. 21 to 24, and SEQ ID NOs. 27 to 36, respectively; the multiplex PCR amplification primer set 4 contains 26 amplification primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 1 to 2, SEQ ID NOs. 7 to 10, SEQ ID NOs. 15 to 18, SEQ ID NOs. 21 to 26, and SEQ ID NOs. NO.37-46; the single-base extension primer set 1 contains 16 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NO.3-4, SEQ ID NO.6 and SEQ ID NO.47-59, respectively; the single-base extension primer set 2 contains 14 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NO.3-6 and SEQ ID NO.60-69, respectively; the single-base extension primer set 3 contains 24 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NO.3-5 and SEQ ID NO.70-90, respectively; the single-base extension primer set 4 contains 19 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NO.3-4 and SEQ ID NO.91-107, respectively.

[0008] Among them, the multiplex PCR amplification primer set 1 and the single base extension primer set 1 are used to detect the resistance of Mycobacterium tuberculosis complex and / or Mycobacterium tuberculosis complex to rifampicin, isoniazid, ethambutol, fluoroquinolones, pyrazinamide, ethionamide and / or prothionamide; the multiplex PCR amplification primer set 2 and the single base extension primer set 2 are used to detect the resistance of Mycobacterium tuberculosis complex and / or Mycobacterium tuberculosis complex to rifampicin, isoniazid, ethambutol, fluoroquinolones, pyrazinamide, rifapentine and / or rifabutin; the multiplex PCR amplification primer set 3 and the single base extension primer set 3 Used to detect the resistance of Mycobacterium tuberculosis complex and / or Mycobacterium tuberculosis complex to rifampicin, isoniazid, ethambutol, fluoroquinolones, streptomycin, capreomycin, kanamycin, amikacin, cycloserine, delamanid, imipenem-cilastatin and / or meropenem; the multiplex PCR amplification primer set 4 and the single base extension primer set 4 are used to detect the resistance of Mycobacterium tuberculosis complex and / or Mycobacterium tuberculosis complex to rifampicin, isoniazid, fluoroquinolones, streptomycin, capreomycin, kanamycin, amikacin, sodium para-aminosalicylate, clofazimine, bedaquiline and / or linezolid.

[0009] Specifically, the Mycobacterium tuberculosis complex identification sites and drug resistance gene sites detected by the multiplex PCR amplification primer set 1 and the single base extension primer set 1 include: IS6110-31, IS6110-45, IS6110-59, rpoB_511-2, rpoB_516-1, rpoB_522-2, rpoB_531-1, rpoB_533-2, rpoB_572-1, inhA_-15, katG_315-1, pncA_57, embB_306-1, embB_406-1, gyrA_91-1 and / or gyrB_539-2; Specifically, the Mycobacterium tuberculosis complex identification sites and drug resistance gene sites detected by the multiplex PCR amplification primer set 2 and the single base extension primer set 2 include: IS6110-31, IS6110-45, IS6110-47, IS6110-59, rpoB_513-1, rpoB_516-3, rpoB_526-1, rpoB_531-3, katG_315-2, pncA_-11, embB_306-3, embB_406-2, gyrA_94-1 and / or gyrB_540-2; Specifically, the Mycobacterium tuberculosis complex identification sites and drug resistance gene sites detected by the multiplex PCR amplification primer set 3 and the single base extension primer set 3 include: IS6110-31, IS6110-45, IS6110-47, rpoB_516-2, rpoB_522-1, rpoB_526-2, rpoB_531-1, katG_315-3, embB_406 -2, gyrA_90-2, gyrB_538-1, gyrB_500-1, gyrB_485-1, rpsL_43-2, rpsL_88-1, rrs_140 1. rrs_1484, alr_261, alr_113, alr_344-3, ald_32, ddn_49-2, ddn_81-2 and / or MarR_42-1; Specifically, the Mycobacterium tuberculosis complex identification sites and drug resistance gene sites detected by the multiple PCR amplification primer group 4 and the single base extension primer group 4 include: IS6110-31, IS6110-45, rpoB_513-2, rpoB_526-3, rpoB_531-3, katG_316-1, gyrA_94-2, gyrA_88-1, gyrB_538-2, gyrB_500-2, rpsL_88-2, rrs_1402, rrs_513 / 514, eis_-14, thyA_202-1, thyA_235-2, folC-43, mmpR5_156-1 and / or rplC_154-1.

[0010] The primer composition of the present invention can be combined with nucleic acid matrix-assisted laser desorption ionization time-of-flight mass spectrometry to detect Mycobacterium tuberculosis complex, and can also detect whether the Mycobacterium tuberculosis complex is resistant to 21 types of commonly used first-line and second-line anti-tuberculosis drugs. Therefore, the present invention also requests protection for the use of the primer composition in the preparation of Mycobacterium tuberculosis complex and / or Mycobacterium tuberculosis complex drug resistance detection products.

[0011] The invention also provides a kit containing the primer combination.

[0012] Specifically, the kit is a kit for detecting Mycobacterium tuberculosis complex and / or detecting the drug resistance of Mycobacterium tuberculosis complex.

[0013] Specifically, the drug resistance is resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, fluoroquinolones, streptomycin, capreomycin, kanamycin, amikacin, ethionamide, prothionamide, sodium para-aminosalicylate, cycloserine, clofazimine, bedaquiline, linezolid, rifapentine, rifabutin, delamanid, imipenem-cilastatin and / or meropenem.

[0014] Optionally, the kit further contains reagents required for multiplex PCR amplification reaction, SAP reaction and / or single base extension reaction.

[0015] Optionally, the kit also contains reagents required for time-of-flight mass spectrometry detection.

[0016] Optionally, the kit also contains reagents required for extracting sample DNA.

[0017] Specifically, the reagents required for the multiplex PCR amplification reaction include PCR reaction buffer and PCR enzyme.

[0018] Specifically, the reagents required for the SAP reaction include SAP reaction buffer and SAP enzyme.

[0019] Specifically, the reagents required for the single base extension reaction include an extension reaction buffer, an extension reaction termination solution and an extension enzyme.

[0020] Specifically, the sample types applicable to the kit of the present invention include: sputum, alveolar lavage fluid, pleural / peritoneal fluid, pus / puncture tissue fluid, cerebrospinal fluid, fresh tissue, urine, blood, feces, strain culture, fresh tissue paraffin sections and wound secretions.

[0021] More specifically, the sample types applicable to the kit include: bacterial strain culture, sputum, alveolar lavage fluid, fresh tissue paraffin sections and wound secretions.

[0022] Specifically, the Mycobacterium tuberculosis complex of the present invention includes Mycobacterium tuberculosis hominis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium voles, Mycobacterium seal, Mycobacterium caprae and Mycobacterium canetti.

[0023] The present invention also provides a method for using the kit, or a method for detecting Mycobacterium tuberculosis complex and / or detecting drug resistance of Mycobacterium tuberculosis complex for non-disease diagnosis purposes, comprising the following steps: S1. Using the genomic DNA of the sample to be tested as a template, using the multiplex PCR amplification primer sets 1 to 4 to perform multiplex PCR amplification reactions respectively, to obtain PCR reaction products 1 to 4; S2. performing SAP reaction on PCR reaction products 1 to 4 respectively to obtain SAP reaction products 1 to 4; S3. Using the obtained SAP reaction products 1 to 4 as templates, mixing them with the single base extension primer sets 1 to 4 in sequence according to their numbers to perform single base extension reactions, thereby obtaining single base extension reaction products 1 to 4; S4. Perform time-of-flight mass spectrometry detection and analysis on the obtained single base extension reaction products and judge the results.

[0024] Specifically, the concentration of each primer in the multiplex PCR amplification primer sets 1 to 4 is 0.1 to 0.5 µM.

[0025] More specifically, each primer is used at a concentration of 0.1 to 0.3 µM.

[0026] Specifically, the concentration of each primer in the single-base extension primer sets 1 to 4 is 0.3 to 2.0 µM.

[0027] More specifically, each primer is used at a concentration of 0.5 to 1.6 µM.

[0028] Specifically, the multiplex PCR amplification reaction program described in S1 is 94-96°C, 2-3 min; 94-96°C, 20-40 sec, 55-57°C, 20-40 sec, 71-73°C, 50-70 sec, 42-45 cycles; 71-73°C, 4-6 min.

[0029] More specifically, the multiplex PCR amplification reaction program is 95°C for 2 min; 95°C for 30 sec, 56°C for 30 sec, 72°C for 60 sec, 45 cycles; 72°C for 5 min.

[0030] Specifically, the SAP reaction procedures in S2 are 36-38 ºC, 35-45 min; 84-86 ºC, 4-7 min.

[0031] More specifically, the SAP reaction procedure is 37 ºC for 40 min; 85 ºC for 5 min.

[0032] Specifically, the single base extension reaction program described in S3 is 94-96°C, 25-35 sec; 38-42 cycles of the following program: ①94-96°C, 4-6 sec, ②51-53°C, 4-6 sec, 79-81°C, 4-6 sec, 5 cycles; 71-73°C, 2-4 min.

[0033] More specifically, the single base extension reaction program is 95°C, 30 sec; 40 cycles of the following program: ①95°C, 5 sec, ②52°C, 5 sec, 80°C, 5 sec, 5 cycles; 72°C, 3 min.

[0034] The present invention has the following beneficial effects: The present invention obtains a set of primer compositions for identifying the target gene IS6110 of the Mycobacterium tuberculosis complex group and genes and mutation sites related to the resistance of the Mycobacterium tuberculosis complex group to 21 types of commonly used first-line and second-line anti-tuberculosis drugs. The primer composition can be used in combination with nucleic acid matrix-assisted laser desorption ionization time-of-flight mass spectrometry to detect the Mycobacterium tuberculosis complex group, and at the same time, the mutation conditions of 18 drug-resistant genes and 92 gene mutations related to the resistance of 21 types of commonly used first-line and second-line anti-tuberculosis drugs in the Mycobacterium tuberculosis complex group can also be detected to guide medication.

[0035] Based on the MassARRAY technology platform, the primer combination has good accuracy and high sensitivity in detecting the Mycobacterium tuberculosis complex and its drug resistance (the minimum detection limit is 10 CFU / mL when the sample is Mycobacterium tuberculosis complex, and the minimum detection limit is 1 ng / μL when the sample is DNA), short time and high efficiency, and is applicable to a variety of sample types, which can meet clinical testing needs, is conducive to the clinical diagnosis and medication guidance of Mycobacterium tuberculosis, and is applicable to a variety of sample types to achieve precise treatment of pulmonary tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the result of PCR primer screening for the target gene site IS6110-31 for identification of Mycobacterium tuberculosis complex; lane M is BM1000 Marker, and the band sizes from top to bottom are 1000 bp, 700 bp, 500 bp, 400 bp, 300 bp, 200 bp, and 100 bp, respectively; lanes 1 to 4 are the amplification products of primer pairs 1 to 4, respectively; lane 5 is water (blank control).

[0037] Figure 2 The results of PCR primer screening for the drug-resistant mutation site rpoB_511-2 of Mycobacterium tuberculosis complex; Lane M is BM1000 Marker, and the band sizes from top to bottom are 1000 bp, 700 bp, 500 bp, 400 bp, 300 bp, 200 bp, and 100 bp, respectively; Lanes 1 and 2 are the amplification products of primer pairs 1 and 2, respectively; Lane 3 is water (blank control).

[0038] Figure 3The results of PCR primer screening for the drug-resistant mutation site katG_315-1 of Mycobacterium tuberculosis complex; Lane M is BM1000 Marker, and the band sizes from top to bottom are 1000 bp, 700 bp, 500 bp, 400 bp, 300 bp, 200 bp, and 100 bp, respectively; Lanes 1 and 2 are the amplification products of primer pairs 1 and 2, respectively; Lane 3 is water (blank control).

[0039] Figure 4 This is the mass spectrometry detection result of the single base extension primer 1 at the rpoB_533-2 site.

[0040] Figure 5 This is the mass spectrometry detection result of the single base extension primer 2 at the rpoB_533-2 site.

[0041] Figure 6 This is the mass spectrometry detection result of pncA_57 and pncA_-11 loci when the detection primers of pncA_57 and pncA_-11 loci are in the same group.

[0042] Figure 7 This is the mass spectrometry detection result of the pncA_57 locus when the detection primers for the pncA_57 and pncA_-11 loci are not in the same group.

[0043] Figure 8 These are the mass spectrometry detection results of multiplex PCR amplification primer set 1 and single base extension primer set 1 for the Mycobacterium tuberculosis quality control product.

[0044] Fig. 9 These are the mass spectrometry detection results of multiplex PCR amplification primer set 2 and single base extension primer set 2 for the Mycobacterium tuberculosis quality control product.

[0045] Fig.10 These are the mass spectrometry detection results of multiplex PCR amplification primer set 3 and single base extension primer set 3 for the quality control product of Mycobacterium tuberculosis.

[0046] Fig.11 These are the mass spectrometry detection results of multiplex PCR amplification primer set 4 and single base extension primer set 4 for the quality control of Mycobacterium tuberculosis.

[0047] Fig.12 The mass spectrometry detection results and Sanger sequencing results of the pncA_57_c.169 C>G site in the example clinical sample. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0049] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0050] Example 1 Obtaining a primer combination based on IS6110 The copy number and position of the gene in the genome of different MTBC strains are highly polymorphic. Combined with the results of the preliminary experiment, the following IS6110 Primers were designed with the IS6110-31, IS6110-45, IS6110-47 and IS6110-59 sites of the gene as targets, and time-of-flight mass spectrometry was used to detect the Mycobacterium tuberculosis complex. In addition, based on the catalog of Mycobacterium tuberculosis resistance genes released by the World Health Organization (WHO), combined with expert consensus, comprehensive consideration of clinical needs and previous experimental exploration results, primers were designed for 18 genes, 59 SNP sites, and 92 gene mutation sites for 21 types of commonly used first-line and second-line anti-tuberculosis drugs, and time-of-flight mass spectrometry was used to detect the drug resistance of Mycobacterium tuberculosis complex.

[0051] The primers designed by the present invention include PCR amplification primers and single-base extension primers, and 2 to 4 pairs of PCR amplification primers and multiple single-base extension primers are designed for each of the sites. The screened suitable PCR amplification primers are formed into a multiplex PCR amplification primer set, and the IS6110-31 site and other drug-resistant mutation sites are amplified simultaneously in the same reaction system, and then the corresponding single-base extension primer set is used to perform a single-base extension reaction. In order to take into account the sensitivity and specificity of the multiplex PCR system, the present invention optimizes the primer set from many aspects in combination with the electrophoresis results of the amplified products. During the optimization process, considering the system pressure, the SNP sites adjacent to the same gene are amplified with the same pair of PCR amplification primers as much as possible; in order to ensure the detection specificity and sensitivity of each site, the primers of each site are optimized; in addition, in order to avoid interference between different primers, the sites are grouped according to the requirement of low mutual influence (not simply divided).

[0052] The process of obtaining the primer composition of the present invention involves multiple aspects. For the convenience of description, some of the processes are selected for exemplary description, as follows: 1. Screening and optimization of PCR amplification primers: obtain primers with good specificity and high amplification efficiency.

[0053] The present invention designs 2 to 4 pairs of PCR amplification primers for each site. Here, the IS6110-31 site, rpoB_511-2 site and katG_315-1 site are taken as examples. The designed primers are shown in Table 1.

[0054] Table 1

[0055] A Cape nucleic acid extraction kit (magnetic bead method DR-4801-KZ type) was used to extract gDNA of Mycobacterium tuberculosis quality control product (purchased from Bondeson Biotechnology Co., Ltd., the quality control product is an inactivated Mycobacterium tuberculosis complex bacterial liquid, and the gDNA extracted from it contains the unmutated SNP sites of 18 genes of the 21 types / classes of commonly used anti-tuberculosis first-line and second-line drugs described in the present invention) and its concentration was measured.

[0056] The extracted genomic DNA of the quality control product was used as a template, and the PCR amplification primers shown in Table 1 were used for PCR amplification reaction. The reaction system was as follows: 2 μL of PCR buffer, 0.1 μL of PCR enzyme, 0.5 μL of primer mixture (the concentration of each primer was 0.1-0.3 μM), 10-50 ng of the quality control product gene DNA, and ultrapure water to make up to 5 μL. The reaction procedure was as follows: (1) 95 °C, 2 min; (2) 95 °C, 30 sec, 56 °C, 30 sec, 72 °C, 60 sec, for a total of 45 cycles; (3) 72 °C, 5 min. After the PCR amplification reaction was completed, the PCR amplification products were detected by electrophoresis.

[0057] The screening results of PCR amplification primers for the IS6110-31 site, rpoB_511-2 site and katG_315-1 site shown in Table 1 are as follows: Figures 1 to 3 As shown. Figure 1 It can be seen that the four pairs of PCR amplification primers for the IS6110-31 locus all have the target amplification bands, but the brightness of the amplification band of the second pair of primers is significantly stronger than that of the first, third, and fourth pairs of primers, indicating that the amplification efficiency of the second pair of primers is the highest, and the second pair of primers is selected for subsequent experiments. Figure 2 It can be seen that the brightness of the amplified band of the second pair of primers at the rpoB_511-2 locus is significantly weaker than that of the first pair of primers, so the first pair of primers was selected for subsequent experiments. Figure 3 It can be seen that both the first and second pairs of PCR amplification primers at the katG_315-1 site have target amplification bands, but the first pair of primers has non-specific amplification, so the second pair of primers was selected for subsequent experiments.

[0058] The same method was used to screen the PCR amplification primers for the remaining sites, and the nucleotide sequences of the PCR amplification primers finally determined are shown in Table 2.

[0059] Table 2 PCR amplification primers

[0060] 2. Screening and optimization of single-base extension primers: obtain extension primers with good specificity and high extension efficiency.

[0061] During the screening and optimization process, the present invention found that some extension primers had low extension efficiency or non-specific extension. In order to improve the detection sensitivity and specificity, in addition to comparative testing of the single-base extension primers at each site, some also redesigned the corresponding single-base extension primers.

[0062] Here, the single base extension primer 1 of the rpoB_533-2 site is used as an example for description. The nucleotide sequence of the primer 1 is: 5'-GGAAGACCGCCGGGCCCC-3'. The process of using it to detect the rpoB_533-2 site is as follows: S1. Using the control gene DNA extracted above as a template, perform PCR amplification using the PCR amplification primers (SEQ ID NO. 7 and 8) for detecting the gene where the rpoB_533-2 site is located (the reaction system and reaction procedure are the same as above) to obtain 5 μL of PCR product; S2. Add 0.17 μL SAP buffer, 0.3 μL SAP enzyme and 1.53 μL ultrapure water to the obtained PCR product (single well) for SAP reaction to obtain 7 μL SAP reaction product; the reaction procedure is 37 ºC 40 min; 85 ºC 5 min; keep warm at 4 ℃; S3. Add 0.2 µL extension buffer, 0.2 µL extension stop solution, 0.15 µL extension enzyme, 0.94 µL single base extension primer (0.5-1.6 µM) and 0.51 µL ultrapure water to the obtained SAP reaction product (single well) for single base extension reaction. After the reaction, centrifuge at 2000 rpm, add 16 µL of sterile double distilled water to each well (the total volume in each well is 25 µL at this time), replace the new membrane to seal the 384-well plate, shake and mix, and centrifuge to obtain the single base extension reaction product; the reaction program is 95 ℃, 30 sec; the following program is repeated for 40 cycles: 1) 95 ℃, 5 sec, 2) 52 ℃, 5 sec, 80 ℃, 5 sec, 5 cycles; 72 ℃, 3 min; keep warm at 4 ℃; S4. Use MassARRAY time-of-flight nucleic acid mass spectrometer to analyze the single-base extension reaction products obtained in S3, import the Assay and the corresponding sample number of the 384-well plate, and connect the chip; place the 384-well plate and the chip in the corresponding positions of the mass spectrometer, press the start button until everything is completed; the test results are automatically analyzed using Typer 4.0 software, and the peak area of ​​each point is exported to an Excel file.

[0063] The mass spectrometry results of the single base extension primer 1 at the rpoB_533-2 site are as follows Figure 4 As shown. Figure 4It can be seen that when the single base extension primer 1 of the rpoB_533-2 site was used for detection, there was no extension product at the rpoB_533-2 site, indicating that the extension efficiency of this single base extension primer was extremely poor. The single base extension primer 2 of the rpoB_533-2 site (5'-AAGCGCCGACTGTCGGCGC-3') was recombinantly designed and detected using the same method as primer 1. The mass spectrometry detection results of the single base extension primer 2 of the rpoB_533-2 site are shown in Figure 2. Figure 5 As shown. Figure 5 It can be seen that there is an extension product of T base at rpoB_533-2 site, which is consistent with the Sanger sequencing result. Therefore, the present invention finally adjusts the single base extension primer sequence of rpoB_533-2 site to 5'-AAGCGCCGACTGTCGGCGC-3'.

[0064] The single-base extension primers for the remaining sites were screened using the same method, and the nucleotide sequences of the single-base extension primers finally determined are shown in Table 3.

[0065] Table 3 Single base extension primer nucleotide sequences

[0066] Table 3

[0067] 3. Primer grouping optimization In order to take into account the sensitivity and specificity of the multiplex PCR system and avoid interference between different primers, the present invention also optimizes the grouping of individual primers.

[0068] First, the primers shown in Tables 2 and 3 were grouped according to the principle that primers with close distribution of the detected SNP sites were divided into different groups, and primers for detecting the same type / class of drug resistance were grouped as much as possible to test their detection effects. Due to interference between primers and other reasons, the detection effect of combining primers with good detection effects in a single system was not satisfactory. For example, the pncA_57 and pncA_-11 sites were originally grouped together, but the test results showed that the pncA_-11 site could produce a normal peak, while the pncA_57 site had a single base extension primer peak without an extension product (such as Figure 6 The pncA_57 site and the pncA_-11 site were separated into different primer sets, and the extension product of the pncA_57 site could be detected normally (as shown in Figure 7 as shown).

[0069] The primer composition finally obtained by the present invention comprises multiplex PCR amplification primer sets 1 to 4 and single base extension primer sets 1 to 4; wherein, multiplex PCR amplification primer set 1 comprises 16 amplification primers shown in SEQ ID NOs. 1 to 2 and SEQ ID NOs. 7 to 20; multiplex PCR amplification primer set 2 comprises 14 amplification primers shown in SEQ ID NOs. 1 to 2, SEQ ID NOs. 7 to 10 and SEQ ID NOs. 13 to 20; multiplex PCR amplification primer set 3 comprises 26 amplification primers shown in SEQ ID NOs. 1 to 2, SEQ ID NOs. 7 to 10, SEQ ID NOs. 13 to 18, SEQ ID NOs. 21 to 24 and SEQ ID NOs. 27 to 36; multiplex PCR amplification primer set 4 comprises 26 amplification primers shown in SEQ ID NOs. 1 to 2, SEQ ID NOs. 7 to 10, SEQ ID NOs. 15 to 18, SEQ ID NOs. 21 to 26 and SEQ ID NOs. 37 to 46; single base extension primer set 1 comprises SEQ ID NOs. NO.3-4, SEQ ID NO.6 and 16 extension primers shown in SEQ ID NO.47-59; single base extension primer set 2 contains 14 extension primers shown in SEQ ID NO.3-6 and SEQ ID NO.60-69; single base extension primer set 3 contains 24 extension primers shown in SEQ ID NO.3-5 and SEQ ID NO.70-90; single base extension primer set 4 contains 19 extension primers shown in SEQ ID NO.3-4 and SEQ ID NO.91-107. The information of genes / sites and nucleotide sequences detected by each PCR amplification primer and single base extension primer is shown in Tables 4-6.

[0070] Table 4 Gene and primer information for detecting Mycobacterium tuberculosis complex

[0071] Table 5 Genes, phenotypes and primer information of first-line drug resistance mutations of Mycobacterium tuberculosis

[0072] Table 6 Second-line drug resistance mutation genes, phenotypes and primer information of Mycobacterium tuberculosis

[0073] Example 2 Kit for detecting Mycobacterium tuberculosis complex and its drug resistance Based on the primer composition described in Example 1, the present invention also provides a kit containing the primer composition and a method of using the kit, which can be used to detect Mycobacterium tuberculosis complex and its drug resistance.

[0074] In addition to the primer composition, the kit also contains the following components: (1) Reagents required for multiplex PCR amplification reaction: including PCR reaction buffer and PCR enzyme; (2) Reagents required for SAP reaction: including SAP reaction buffer and SAP enzyme; (3) Reagents required for single base extension reaction: including extension reaction buffer, extension reaction termination solution and extension enzyme; (4) Reagents required for time-of-flight mass spectrometry detection: specifically, reagents required for nucleic acid matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection.

[0075] The method for detecting Mycobacterium tuberculosis complex and its drug resistance using the kit is the same as in Example 1, specifically as follows: S1. Using the genomic DNA of the sample to be tested as a template, using the multiplex PCR amplification primer sets 1 to 4 to perform multiplex PCR amplification reactions respectively, to obtain PCR reaction products 1 to 4; S2. performing SAP reaction on PCR reaction products 1 to 4 respectively to obtain SAP reaction products 1 to 4; S3. Using the obtained SAP reaction products 1 to 4 as templates, mixing them with the single base extension primer sets 1 to 4 in sequence according to their numbers to perform single base extension reactions, thereby obtaining single base extension reaction products 1 to 4; S4. Perform time-of-flight mass spectrometry detection and analysis on the obtained single base extension reaction products and judge the results.

[0076] The reaction system (single-well dosage) of the multiplex PCR amplification reaction in S1 was: 2 µL of PCR buffer, 0.1 µL of PCR enzyme, 0.5 µL of amplification primer mixture (the concentration of each primer in the mixture was 0.1-0.3 µM), 10-50 ng of DNA, and ultrapure water to make up to 5 µL; the reaction program was: 95 °C for 2 min; 95 °C for 30 sec, 56 °C for 30 sec, 72 °C for 60 sec, for a total of 45 cycles; 72 °C for 5 min; and insulation at 4 °C.

[0077] The reaction system (single-well dosage) of the SAP reaction in S2 is: 1.53 μL ultrapure water, 0.17 μL SAP buffer, 0.3 μL SAP enzyme, 5 μL of the multiplex PCR reaction product obtained in S1, total volume: 7 μL; the reaction program is: 37 ºC 40 min; 85 ºC 5 min; insulation at 4 ℃.

[0078] The reaction system (single-well dosage) of the single-base extension reaction in S3 is: 0.51 µL ultrapure water, 0.2 µL extension buffer, 0.2 µL extension stop solution, 0.15 µL extension enzyme, 0.94 µL single-base extension primer mixture (the concentration of each primer in the mixture is 0.5-1.6 µM), and 7 µL of SAP reaction product obtained in S2; the reaction program is: 95 °C, 30 sec; 40 cycles of the following program: 1) 95 °C, 5 sec, 2) 52 °C, 5 sec, 80 °C, 5 sec, 5 cycles; 72 °C, 3 min; 4 °C insulation; after the reaction, centrifuge at 2000 rpm, add 16 µL of sterile double-distilled water to each well (the total volume in each well is 25 µL at this time), replace a new membrane to seal the 384-well plate, shake and mix, and centrifuge to obtain the single-base extension reaction product.

[0079] The result judgment method in S4 is: perform time-of-flight mass spectrometry analysis on the obtained single base extension reaction product, detect the flight time of the extension reaction product in the vacuum tube, and calculate the molecular weight of the extension reaction product; then compare the molecular weight of the extension reaction product with the preset molecular weight in the software through the analysis software, and judge the peak of the extension reaction product as wild type, heterozygous mutant type or homozygous mutant type. The molecular weight of the extension primer and extension product of each type at each site is shown in Table 7. According to different test results, judge whether the sample is infected with Mycobacterium tuberculosis and whether there is a drug-resistant phenotype.

[0080] Table 7 Correspondence between the molecular weight of each type of extension primer and extension product at each site

[0081] Table 7

[0082] Table 7

[0083] The present invention uses the extracted Mycobacterium tuberculosis quality control product DNA as a template and uses the kit to detect it. The mass spectrometry detection results obtained by using multiple PCR amplification primer sets 1 to 4 and single base extension primer sets 1 to 4 are as follows: Figures 8 to 11 As shown. Figures 8 to 11 It can be seen that the corresponding results can be read out according to the molecular weight of the extension product at each site, that is, the kit can be used to accurately detect 92 gene mutation sites of the Mycobacterium tuberculosis complex and 18 drug-resistant genes related to the resistance of the Mycobacterium tuberculosis complex to 21 types of commonly used first-line and second-line anti-tuberculosis drugs, indicating that the kit of the present invention can be used to detect the resistance of the Mycobacterium tuberculosis complex to 21 types of commonly used first-line and second-line anti-tuberculosis drugs to guide medication.

[0084] Example 3 Detection limit determination The present invention determines the minimum detection limit of the Mycobacterium tuberculosis complex liquid or its DNA by the kit described in Example 2. The minimum detection limit of DNA is based on the plasmid synthesized by Sangon Biotech (Shanghai) Co., Ltd. as a template.

[0085] The details are as follows: 1. Using the quality control product as a template, determine the minimum detection limit: The initial concentration of the Mycobacterium tuberculosis quality control product is 10000 CFU / mL, and it is diluted in gradients to dilute the quality control product concentrations to 1000 CFU / mL, 100 CFU / mL, 50 CFU / mL, 10 CFU / mL and 5 CFU / mL. Referring to the method described in Example 2, the DNA of each diluted Mycobacterium tuberculosis quality control product is extracted respectively, and the kit described in Example 2 is used to perform multiple PCR amplification reaction, SAP digestion, single base extension reaction and mass spectrometry detection analysis. Each gradient is tested 3 times; the lowest concentration that can be detected 100% is taken as the minimum detection limit of the product, and then at this concentration, the test is repeated 5 times, and all 5 times are detected, then the concentration can be determined as the minimum detection limit of the product. Finally, it is determined that the minimum detection limit of the kit of the present invention for the Mycobacterium tuberculosis complex bacterial liquid is 10 CFU / mL.

[0086] 2. Using plasmid as template, determine the minimum detection limit: The wild type and the mutant plasmids of all sites involved in the detection of the present invention (i.e., the plasmids containing the 18 drug resistance genes and the plasmids containing the 92 mutations) were mixed together, totaling 110 types, a total of 4 μg plasmids, and 40 μL of ultrapure water or TE was added and mixed thoroughly to obtain a plasmid solution with an initial concentration of 100 ng / μL, which was gradiently diluted to 10 ng / μL, 5ng / μL, 2 ng / μL and 1 ng / μL. Referring to the experimental steps of Example 2, multiple PCR amplification reaction, SAP digestion, single base extension reaction and mass spectrometry detection analysis were performed respectively. Each gradient was tested 3 times; the lowest concentration that could be detected 100% was taken as the minimum detection limit of the product, and then at this concentration, the test was repeated 5 times, and all 5 times were detected, then the concentration was determined to be the minimum detection limit of the product. Finally, the minimum detection limit of the present invention was determined to be 1 ng / μL.

[0087] Example 4 Testing of different sample types In actual clinical testing, the types of samples collected by different departments will be different. In order to test the detection of different types of samples by the kit described in the present invention, 5 different types of clinical samples were collected and tested using the kit and method described in Example 2. The 5 different types of clinical samples are strain cultures, sputum, alveolar lavage fluid, fresh tissue paraffin sections, and wound secretions; each sample type contains 5 clinical samples, for a total of 25 clinical samples. In addition to performing mass spectrometry detection on the 25 clinical samples using the kit described in the present invention, the 25 clinical samples were also tested by Sanger sequencing, and the accuracy of the mass spectrometry detection was evaluated by comparing the mass spectrometry detection results with the Sanger sequencing results.

[0088] The comparison of the mass spectrometry detection results and the Sanger sequencing results of the 25 clinical samples is shown in Table 8. As can be seen from Table 8, the mass spectrometry detection results of the clinical samples are completely consistent with the Sanger sequencing results.

[0089] Table 8 Comparison of mass spectrometry results and Sanger sequencing results of different clinical samples

[0090] Table 8

[0091] As an example, the present invention provides the mass spectrometry detection results and Sanger sequencing results of the pncA_57 site of one of the clinical samples, such as Fig.12 As shown. Fig.12 It can be seen that the mass spectrometry detection results are consistent with the Sanger sequencing results.

[0092] Using Sanger sequencing as the gold standard, the total coincidence rate of the mass spectrometry detection (MassARRAY) results of the above 25 clinical samples was statistically analyzed, and the results are shown in Table 9. As shown in Table 9, the coincidence rate of sensitive type is 100%, the coincidence rate of drug-resistant type is 100%, the coincidence rate of tuberculosis negative is 100%, and the total coincidence rate (total accuracy) is 100%.

[0093] Table 9 Statistics of MassARRAY and Sanger sequencing test results

[0094] From the results shown in Tables 8 and 9, it can be seen that the kit of the present invention can accurately detect 92 gene mutation sites of 18 drug-resistant genes related to the resistance of Mycobacterium tuberculosis complex to 21 types / classes of commonly used first-line and second-line anti-tuberculosis drugs, with high detection accuracy.

[0095] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A primer composition, characterized in that It comprises multiplex PCR amplification primer sets 1 to 4 and single base extension primer sets 1 to 4; The multiplex PCR amplification primer set 1 contains 16 amplification primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 1 to 2 and SEQ ID NOs. 7 to 20 respectively; The multiplex PCR amplification primer set 2 contains 14 amplification primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 1 to 2, SEQ ID NOs. 7 to 10, and SEQ ID NOs. 13 to 20, respectively; The multiplex PCR amplification primer set 3 contains 26 amplification primers, and the nucleotide sequences of the primers are shown as SEQ ID NOs. 1 to 2, SEQ ID NOs. 7 to 10, SEQ ID NOs. 13 to 18, SEQ ID NOs. 21 to 24, and SEQ ID NOs. 27 to 36, respectively; The multiplex PCR amplification primer set 4 contains 26 amplification primers, and the nucleotide sequences of the primers are shown as SEQ ID NOs. 1 to 2, SEQ ID NOs. 7 to 10, SEQ ID NOs. 15 to 18, SEQ ID NOs. 21 to 26, and SEQ ID NOs. 37 to 46, respectively; The single-base extension primer set 1 contains 16 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 3 to 4, SEQ ID NO. 6, and SEQ ID NOs. 47 to 59, respectively; The single-base extension primer set 2 contains 14 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 3 to 6 and SEQ ID NOs. 60 to 69 respectively; The single-base extension primer set 3 contains 24 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 3 to 5 and SEQ ID NOs. 70 to 90 respectively; The single-base extension primer set 4 contains 19 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 3 to 4 and SEQ ID NOs. 91 to 107, respectively.

2. Use of the primer composition according to claim 1 in the preparation of a Mycobacterium tuberculosis complex and / or a Mycobacterium tuberculosis complex drug resistance detection product, characterized in that: The drug resistance is resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, fluoroquinolones, streptomycin, capreomycin, kanamycin, amikacin, ethionamide, prothionamide, sodium para-aminosalicylate, cycloserine, clofazimine, bedaquiline, linezolid, rifapentine, rifabutin, delamanid, imipenem-cilastatin and / or meropenem.

3. A kit comprising the primer combination according to claim 1.

4. The kit according to claim 3, characterized in that: The kit is a kit for detecting Mycobacterium tuberculosis complex and / or detecting the drug resistance of Mycobacterium tuberculosis complex; the drug resistance is resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, fluoroquinolones, streptomycin, capreomycin, kanamycin, amikacin, ethionamide, prothionamide, sodium para-aminosalicylate, cycloserine, clofazimine, bedaquiline, linezolid, rifapentine, rifabutin, delamanid, imipenem-cilastatin and / or meropenem.

5. The kit according to claim 4, characterized in that: The kit also contains reagents required for multiplex PCR amplification reaction, SAP reaction and / or single base extension reaction.

6. The kit according to claim 4, characterized in that: The kit also contains reagents required for time-of-flight mass spectrometry detection.

7. The kit according to claim 4, characterized in that: The kit also contains reagents required for extracting sample DNA.

8. The kit according to claim 5, characterized in that: The reagents required for the multiplex PCR amplification reaction include PCR reaction buffer and PCR enzyme.

9. The kit according to claim 5, characterized in that The reagents required for the SAP reaction include SAP reaction buffer and SAP enzyme.

10. The kit according to claim 5, characterized in that: The reagents required for the single base extension reaction include an extension reaction buffer, an extension reaction termination solution and an extension enzyme.

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