A kit and use thereof

Abstract

The present disclosure provides a kit and its application, the kit comprising two sets of amplification primer groups, single base extension primers and processing reagents; the amplification primer groups are used for amplifying Mycobacterium tuberculosis flora identification sites and anti-tuberculosis drug pharmacogenetic polymorphism sites; each pair of primers corresponds to the upstream and downstream regions of a SNP site; the processing reagents include PCR reaction mixture, PCR enzyme mixture, SAP reaction mixture, SAP enzyme mixture, extension reaction mixture and extension enzyme mixture. By designing specific amplification primer groups and single base extension primers, the system and reagents are optimized, and the sensitivity is high. The kit combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry can complete the detection of all sites, and the detection cost is low and the efficiency is high.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of molecular diagnosis, in particular to a kit and application thereof. BACKGROUND

[0002] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (MTB), and according to the World Health Organization, about 2 billion people in the world are infected with Mycobacterium tuberculosis, and about 3 million people die of tuberculosis every year. The drug resistance of Mycobacterium tuberculosis to anti-tuberculosis drugs further aggravates the prevalence and spread of tuberculosis in a large range. Drug-resistant Mycobacterium tuberculosis, especially multi-drug resistant Mycobacterium tuberculosis (MDR-TB), spreads rapidly, and the patient's condition progresses rapidly, with a high mortality rate. Some cases from diagnosis to death are only 4-6 weeks.

[0003] In general clinical laboratories, Mycobacterium tuberculosis drug sensitivity detection still uses traditional methods such as absolute concentration method, ratio method and resistance ratio method. Since Mycobacterium tuberculosis grows slowly, it still takes 1-2 months to obtain results after obtaining the isolated culture, so relying on traditional bacterial culture and drug sensitivity test will delay valuable treatment time. Although the BACTECTM MGITTM 960 system developed in recent years can shorten the period of Mycobacterium tuberculosis culture and drug sensitivity test, it still takes about a week. Moreover, the instrument equipment and reagents are quite expensive, so it can only be carried out in a few hospitals. Numerous studies have shown that rapid nucleic acid detection of Mycobacterium tuberculosis pathogens is more sensitive than traditional sputum smear and Mycobacterium tuberculosis culture detection, and is more conducive to early clinical diagnosis.

[0004] Currently, as a medium-throughput technology in the field of gene detection, Matrix Assisted Laser Desorption / Ionization-Time of Flight (MALDI-TOF) has a wide detection range. However, the existing kit can only detect one gene / polymorphism type of gene at a time, and the specificity of the amplification primer is not high, so a kit based on Matrix Assisted Laser Desorption / Ionization-Time of Flight (MALDI-TOF) platform for Mycobacterium tuberculosis flora identification and drug-resistant mutation site detection is needed. SUMMARY

[0005] The present disclosure provides a kit and application thereof to at least solve the problems of low specificity of amplification primer and low detection efficiency in the prior art.

[0006] According to a first aspect of the present disclosure, a kit is provided, which comprises two sets of amplification primer groups, a single base extension primer, and processing reagents;

[0007] The amplification primer groups are used to amplify Mycobacterium tuberculosis population identification sites, including IS6110, rdl, rd4, rd9, ext-rd9, rd12, human reference gene sites IC2_1, IC2_2; the amplification primer groups are also used to amplify anti-tuberculosis drug pharmacogenetic polymorphism sites, including rrs905_2, pncA11, inhA15_2, gyrB499_2, rrs905_1, pncA76, katG315_2, rpoB516_2, rrs513_1, gyrB499_1, rpsL43_1, rpoB516_1, gyrA90_1, inhA15_1, rpoB533, katG315_1, rpoB531, gyrA94_1, embB306_1, rpoB526_2, rpsL43_2, rpsL88_1, rpoB511, rrs513_2, gyrA94_2, embB306_2, rpoB526_1, rpsL88_2; each pair of primers corresponds to the upstream and downstream regions of a SNP site, respectively.

[0008] The processing reagents include PCR reaction mix, PCR enzyme mix, SAP reaction mix, SAP enzyme mix, extension reaction mix, and extension enzyme mix.

[0009] In an implementation, the two sets of amplification primer groups are a first set of amplification primer groups and a second set of amplification primer groups, wherein,

[0010] The first set of amplification primer groups includes detection sites: rrs905_2, rd4, IC2_1, ext-rd9, rdl, pncA11, inhA15_2, gyrB499_2, rrs905_1, pncA76, katG315_2, rpoB516_2, rrs513_1, gyrB499_1, rpsL43_1, rpoB516_1, gyrA90_1, inhA15_1, IS6110, rpoB533, rd12, katG315_1, rpoB531.

[0011] The second set of amplification primer groups includes detection sites: gyrA94_1, IC2_2, embB306_1, rd9, rpoB526_2, rpsL43_2, rpsL88_1, rpoB511, rrs513_2, gyrA94_2, embB306_2, rpoB526_1, rpsL88_2.

[0012] In an implementation, the first set of amplification primer groups include amplification primer sequences of detection sites as shown in SEQ ID NO. 1-46, respectively; and the second set of amplification primer groups include amplification primer sequences of detection sites as shown in SEQ ID NO. 47-72, respectively.

[0013] In an implementation, the primer sequences of the single base extension primers are as shown in SEQ ID NO. 73-108, respectively.

[0014] In an implementation, the sequences shown in SEQ ID NO. 96, SEQ ID NO. 103, and SEQ ID NO. 107 are subjected to degenerate base, 5' thio hydrolysis-resistant modification, 3' MGB modification, and the molecular weights of thio and MGB are 328.4 g / mol and 1120.6 g / mol, respectively.

[0015] In an implementation, the processing reagent further includes an amplification reaction primer premix, and the amplification reaction primer premix is a mixture of nucleotide sequences shown in SEQ ID NO. 1-72, and the concentration of each amplification primer is 0.3-3 µM.

[0016] In an implementation, the processing reagent further includes a single base extension reaction primer premix, and the single base extension reaction primer premix is a mixture of nucleotide sequences shown in SEQ ID NO. 73-108, and the concentration of each single base extension primer is 5-30 µM.

[0017] In an implementation, the kit further includes a desalting resin and a detection chip, and the detection chip includes a silicon-based chip containing 384 detection points with pre-pointed matrix.

[0018] According to a second aspect of the present disclosure, a kit is provided for use in Mycobacterium tuberculosis population identification and drug-resistant mutation site detection.

[0019] According to a third aspect of the present disclosure, an integrated nucleic acid mass spectrometry detection system is provided, including a matrix-assisted laser desorption ionization time-of-flight mass spectrometry system and the kit of the present disclosure.

[0020] The kit and its application provided by the present disclosure can achieve excellent specificity of amplification primer groups and single base extension primers, high sensitivity of optimized system and reagents, and complete site detection by combining the kit with matrix-assisted laser desorption ionization time-of-flight mass spectrometry, so as to obtain all Mycobacterium tuberculosis species information and drug-resistant mutation site information at a relatively low cost, and avoid the problems of high cost and low efficiency caused by repeated detection.

[0021] It is to be understood that the details set forth herein are by way of example and not intended to limit the scope of the subject disclosure. Other features and characteristics of the subject disclosure will become more fully apparent from the following detailed description and appended claims. BRIEF DESCRIPTION OF DRAWINGS

[0022] The above and other objects, features and advantages of the present exemplary embodiments will become more fully apparent from the following detailed description, the appended claims and the accompanying drawings. In the drawings, the reference numerals

[0023] In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals.

[0024] 1 in Fig. 1 .1-1.4 is the detection result of sample No. 1 by the first group of primers, the software interface of the DP-TOF nucleic acid mass spectrometer, the software automatically interprets the base type of each site according to the peak of the product peak after the extension of each hole;

[0025] 2 in Fig. 2 .1-2.4 is the detection result of sample No. 2 by the first group of primers, the software interface of the DP-TOF nucleic acid mass spectrometer, the software automatically interprets the base type of each site according to the peak of the product peak after the extension of each hole;

[0026] 3 in Fig. 3 .1-3.4 is the detection result of sample No. 1 by the second group of primers, the software interface of the DP-TOF nucleic acid mass spectrometer, the software automatically interprets the base type of each site according to the peak of the product peak after the extension of each hole;

[0027] 4 in Fig. 4 .1-4.4 is the detection result of sample No. 2 by the second group of primers, the software interface of the DP-TOF nucleic acid mass spectrometer, the software automatically interprets the base type of each site according to the peak of the product peak after the extension of each hole;

[0028] Fig. 5 Sanger sequencing peak chart of strain 1 (mutation exists in the gyrA94_1 extension primer segment);

[0029] Fig. 6 Mass spectrometry detection peak chart of strain 1 (mutation exists in the gyrA94_1 extension primer segment);

[0030] Fig. 7 Sanger sequencing peak chart of strain 2 (mutation exists in the rpoB511 extension primer segment);

[0031] Fig. 8 Mass spectrometry detection peak chart of strain 2 (mutation exists in the rpoB511 extension primer segment);

[0032] Fig. 9 Sanger sequencing peak diagram of strain 3 (with a mutation in the extension primer region of rpoB526_1);

[0033] Fig. 10 This is a mass spectrometry peak diagram of strain 3 (rpoB526_1 extension primer segment has mutation). Detailed Implementation

[0034] To make the objectives, features, and advantages of this disclosure more apparent and understandable, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0035] Example 1

[0036] This invention provides a kit comprising two sets of amplification primers, single-base extension primers, and processing reagents;

[0037] The primer set was used to amplify identification sites of Mycobacterium tuberculosis, including IS6110, rd1, rd4, rd9, ext-rd9, rd12, and human internal reference gene sites IC2_1 and IC2_2. The primer set was also used to amplify pharmacogene polymorphism sites of anti-tuberculosis drugs, including rrs905_2, pncA11, inhA15_2, gyrB499_2, rrs905_1, pncA76, katG315_2, rpoB516_2, rrs513_1, and gyrB499. _1, rpsL43_1, rpoB516_1, gyrA90_1, inhA15_1, rpoB533, katG315_1, rpoB531, gyrA94_1, embB306_1, rpoB526_2, rpsL43_2, rpsL88_1, rpoB511, rrs513_2, gyrA94_2, embB306_2, rpoB526_1, rpsL88_2; Each primer pair corresponds to the upstream and downstream regions of a SNP site.

[0038] The processing reagents include PCR reaction mixture, PCR enzyme mixture, SAP reaction mixture, SAP enzyme mixture, extension reaction mixture, and extension enzyme mixture.

[0039] The two sets of amplification primers are the first set and the second set, respectively.

[0040] The first group of amplification primer sets includes detection sites: rrs905_2, rd4, IC2_1, ext-rd9, rd1, pncA11, inhA15_2, gyrB499_2, rrs905_1, pncA76, katG315_2, rpoB516_2, rrs513_1, gyrB499_1, rpsL43_1, rpoB516_1, gyrA90_1, inhA15_1, IS6110, rpoB533, rd12, katG315_1, rpoB531;

[0041] The second group of amplification primer sets includes detection sites: gyrA94_1, IC2_2, embB306_1, rd9, rpoB526_2, rpsL43_2, rpsL88_1, rpoB511, rrs513_2, gyrA94_2, embB306_2, rpoB526_1, rpsL88_2.

[0042] The amplification primers and single base extension primers described above are synthesized by Shanghai Bailige Biotechnology Co., Ltd., and the sequences are shown in Tables 1 and 2.

[0043] Table 1 Amplification primer sequence

[0044]

[0045]

[0046] Table 2 Single base extension primer sequence

[0047]

[0048]

[0049] In one example, the sequences shown in SEQ ID NO. 96, SEQ ID NO. 103, and SEQ ID NO. 107 are subjected to degenerate bases, 5' thio hydrolysis-resistant modification, and 3' MGB modification. The molecular weights of thio and MGB are 328.4 g / mol and 1120.6 g / mol, respectively.

[0050] In one example, the processing reagents include PCR reaction mixture, PCR enzyme mixture, SAP reaction mixture, SAP enzyme mixture, extension reaction mixture, and extension enzyme mixture.

[0051] The PCR reaction mixture includes: PCR buffer, Mg 2+ , dATP, dCTP, dTTP, dUTP, and dGTP, etc.

[0052] PCR enzyme mixture includes Taq enzyme and UNG enzyme;

[0053] SAP reaction mixture includes SAP buffer solution;

[0054] SAP enzyme mixture includes SAP enzyme and enzyme preservative solution;

[0055] Extension reaction mixture includes extension buffer solution, Mg 2+ , ddATP, ddCTP, ddTTP, ddGTP, etc.

[0056] Extension enzyme mixture includes extension enzyme, enzyme preservative solution and glycerol.

[0057] In one example, the processing reagent further includes an amplification reaction primer premix, and the amplification reaction primer premix is a mixture of nucleotide sequences shown in the sequence listing of SEQ ID NO. 1-72, and each amplification primer has a concentration of 0.3-3 μM.

[0058] In one example, the processing reagent further includes a single base extension reaction primer premix, and the single base extension reaction primer premix is a mixture of nucleotide sequences shown in the sequence listing of SEQ ID NO. 73-108, and each single base extension primer has a concentration of 5-30 μM.

[0059] In one example, the kit further includes:

[0060] Salt removal resin: including cation exchange resin powder for removing salt ions in the extension reaction solution;

[0061] Detection chip: including a silicon-based chip containing 384 pre-spotted substrates;

[0062] Pure mutant control: including a water solution of 21-site mutant Mycobacterium tuberculosis drug resistance gene corresponding fragment plasmids diluted after absolute quantification by fluorescence quantitative standard curve method, with a concentration of more than 1000 copies / μl;

[0063] Hybrid control: including a mixed water solution of 21-site mutant and wild type Mycobacterium tuberculosis drug resistance gene corresponding fragment plasmids diluted after absolute quantification by fluorescence quantitative standard curve method, with a concentration of more than 1000 copies / μl;

[0064] Pure wild control: including a water solution of 21 wild type Mycobacterium tuberculosis drug resistance gene corresponding fragment plasmids diluted after absolute quantification by fluorescence quantitative standard curve method, with a concentration of more than 1000 copies / μl.

[0065] The kit provided by this invention is used for the identification of Mycobacterium tuberculosis flora and the detection of drug resistance mutation sites. By designing a highly specific set of amplification primers and single-base extension primers, the optimized system and reagents have high sensitivity. By combining this kit with matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, all sites can be detected. This allows for the acquisition of information on all Mycobacterium tuberculosis species and drug resistance mutation sites in a single test at a low cost, avoiding the high cost and low efficiency caused by repeated testing.

[0066] Example 2

[0067] A method for using a kit for identifying Mycobacterium tuberculosis flora and detecting drug-resistant mutation sites, wherein the kit is the kit described in this invention; the method includes the following steps:

[0068] S1. Prepare Mycobacterium tuberculosis DNA template;

[0069] First, samples are collected. Sample types can include patient sputum, bronchoalveolar lavage fluid, bacterial culture medium, cotton swabs, body fluids, and feces. Second, bacterial genomic DNA is extracted from the collected samples. In this embodiment, bacterial genomic DNA is extracted from the collected samples using the Tianlong Technology Ex-DNA Bacterial Genome Extraction (Special Extraction, Cat.T132) extraction manual.

[0070] S2. Detect the extracted bacterial genomic DNA using two sets of amplification primers and two sets of single-base extension primers;

[0071] Step S2 includes:

[0072] S21. Prepare PCR reagents;

[0073] Take the PCR reaction mixture and PCR enzyme mixture from the kit, thaw them at room temperature and mix them by shaking. Centrifuge at 2000 rpm for 10 seconds and calculate the number of reaction reagents required.

[0074] The PCR test reaction system includes: 1.67 μL of PCR reaction mixture, 0.33 μL of PCR enzyme mixture, and 1 μL of PCR amplification primer premix.

[0075] Based on the PCR test reaction system, calculate the amount of each reagent used, mix thoroughly, aliquot 3 μL into PCR reaction tubes or 384-well PCR plates, and transfer to the sample processing area.

[0076] S22, Sample loading;

[0077] Add 2 μL of the bacterial genomic DNA solution extracted in step S1, seal the reaction tube or 384-well PCR plate tightly, and then transfer it to the detection area.

[0078] S23, PCR amplification reaction;

[0079] Put each reaction tube in a certain order on the PCR instrument, and perform PCR amplification according to the following program:

[0080]

[0081] The PCR product is temporarily not subjected to the next experimental operation, and can be stored at 4°C overnight.

[0082] Under the guidance of the primer, the DNA template (i.e. the extracted bacterial genomic DNA) sequentially undergoes pre-denaturation, denaturation, extension, and annealing to complete the PCR amplification reaction.

[0083] S24, preparation of SAP reaction solution;

[0084] Take out the SAP reaction mixture and SAP enzyme mixture from the kit, melt and shake well at room temperature, and then centrifuge at 2000 rpm for 10 sec. Calculate the number of reaction reagents needed.

[0085] Each test reaction system includes: SAP reaction mixture 1.70 μL, SAP enzyme mixture 0.30 μL.

[0086] S25, sample loading (nucleic acid amplification zone);

[0087] Add 2 μL of the above SAP reaction solution to the PCR amplification product of S23, and tightly cover the reaction tube or 384-well PCR plate.

[0088] Put each reaction tube in a certain order on the PCR instrument, and perform SAP digestion according to the following program:

[0089]

[0090]

[0091] The SAP product should be immediately subjected to the next operation, and it is not recommended to be placed at 4°C overnight.

[0092] S26, preparation of extension reagent;

[0093] Take out the extension reaction mixture, extension enzyme mixture, and single base extension reaction premix from the kit, melt and shake well at room temperature, and then centrifuge at 2000 rpm for 10 sec. Calculate the number of reaction reagents needed.

[0094] Each test reaction system includes: extension reaction mixture 0.72 μL, extension enzyme mixture 0.34 μL, single base extension reaction premix 0.94 μL.

[0095] S27, adding sample (nucleic acid amplification area);

[0096] Add 2 μL of the above-mentioned extension reaction solution to the SAP product of S26 in sequence, and tightly cover the reaction tube or 384-hole PCR plate.

[0097] Place each reaction tube on the PCR instrument in sequence, and perform extension amplification according to the following procedure:

[0098]

[0099] If the extension product is not subjected to the next step of experimental operation, it can be stored at 4°C overnight.

[0100] S28, mass spectrometry detection using a DP-TOF time-of-flight mass spectrometry detection system (amplification analysis area):

[0101] According to the operation manual of the DP-TOF nucleic acid mass spectrometer, select the required chip and hole number corresponding to the sample to be detected, and the instrument automatically performs sample extension product desalination, chip spotting and detection, and automatically analyzes the results.

[0102] S29, import the result file analyzed by the mass spectrometer into the sample result report system, and issue the sample result report.

[0103] For example Figs. 1-4 , the detection results of sample No. 1 and No. 2 using the first group of primers and the second group of primers are shown. Taking FIG. 1 as an example, Fig. 1.1 , a schematic diagram for illustrating the detection result of sample 1 using the first group of primers, the circular "O" represents an unknown sample, and the diamond "◇" represents a blank control; dark green represents the best detection result, light green represents the second best detection result, yellow represents the third best detection result, and red represents the worst detection result. Since the mass spectrometry detection result page is colored, and the drawings of the present application are black and white, the drawings of the present application distinguish the dark green, light green, yellow and red of the mass spectrometry software interface by color depth, wherein red is the darkest color, dark green is the second darkest color, yellow is the third darkest color, and light green is the lightest color. There is no yellow in the present application Fig. 1.1 , for example Fig. 1.1 , the M row 18th column and 20th column and the P row 20th column and 24th column are red; the M row 22nd column, the N row 19th column, the O row 20th column and 23rd column, and the P row 18th column are light green, and the rest are dark green. Fig. 1.2 , and Fig. 1.3 , are the peak out-of-peak situations of the post-extension product of each hole, Fig. 1.4 , is the automatic interpretation of the base type of each site by the system.

[0104] Example 3

[0105] The accuracy test of the detection of the drug-resistant mutation site of Mycobacterium tuberculosis using the kit described in the present application is as follows:

[0106] (1) Extraction of 10 known positive clinical sputum samples of Mycobacterium tuberculosis for tuberculosis genomic DNA extraction;

[0107] (2) Detection of 10 sputum samples of tuberculosis according to Example 2 using 2 groups of primers;

[0108] (3) The Sanger sequencing method was used to verify the detection accuracy of the kit, and the verification primer pairs for amplifying the regions of each site were synthesized by Shanghai Bailige Biotechnology Co., Ltd., and the sequences are shown in Table 3.

[0109] Table 3 Corresponding site sequences

[0110]

[0111]

[0112] (4) The verification PCR reaction system is as follows:

[0113]

[0114] (5) Mix the reaction mixture, centrifuge at 8000g, and perform verification PCR, the program is as follows:

[0115]

[0116]

[0117] (6) After each sample PCR amplification, 2% agarose electrophoresis is used to determine the amplified fragments, and the sequencing company (Hangzhou Shangya Biotechnology Co., Ltd.) is used for sequencing verification, and the results are compared with the mass spectrometry detection results. Take one example, the comparison results are shown in the following Table 4.

[0118] Table 4 Comparison results

[0119]

[0120]

[0121] Conclusion: The Sanger sequencing method detection results are completely consistent with the nucleic acid flight mass spectrometry results.

[0122] Advantages of the present application: 2-hole reaction detects all drug-resistant mutation sites, while the traditional sequencing method needs 13-hole reaction, and the workload and cost are much higher than the method.

[0123] Example 4

[0124] The kit is used for Mycobacterium tuberculosis population identification and drug-resistant mutation site detection sensitivity test.

[0125] In this embodiment, two DNA samples extracted from the National Reference Standard for Mycobacterium tuberculosis by the National Institutes for Food and Drug Control were selected and serially diluted. The initial DNA concentration was determined using a Qubit spectrophotometer. The initial DNA loading amounts were 0.5 ng / μl, 0.1 ng / μl, 0.05 ng / μl, 0.025 ng / μl, and 0.001 ng / μl, respectively. Subsequent PCR, digestion, extension, and mass spectrometry were performed. The results of the two samples are shown in Tables 5-6 below.

[0126] Table 5 Sensitivity test results for Sample 1

[0127]

[0128]

[0129] Table 6. Sensitivity test results for Sample 2

[0130]

[0131]

[0132] The detection results in Tables 5 and 6 show that when the sample loading amount was as low as 0.05 ng / μl, all sites in both samples were correctly detected. However, when the loading amount was 0.025 ng / μl, only a few sites were detected. This indicates that this kit can detect all sites simultaneously with bacterial genomic DNA at a concentration as low as 0.05 ng / μl, demonstrating very high sensitivity.

[0133] Example 5

[0134] The kit described in this invention can detect strain variants with base mutations in extended primer regions through degenerate primer design.

[0135] This embodiment selected three Mycobacterium tuberculosis strains whose extended primer regions were confirmed by Sanger sequencing to have base mutations: strain 1 (mutation in the gyrA94_1 extended primer region), strain 2 (mutation in the rpoB511 extended primer region), and strain 3 (mutation in the rpoB526_1 extended primer region). The kit described in this invention was used for detection, and the results are shown in the attached comparison. Figs. 5-10 As shown.

[0136] In the kit described in this invention, primers SEQ ID NO.96, SEQ ID NO.103, and SEQ ID NO.107 are designed with degenerate bases. The results show that this kit can also detect the corresponding sites for strains with base mutations in the extended primer segments, making it suitable for the study of complex or rare samples.

[0137] In another aspect of the present application, there is provided an integrated nucleic acid mass spectrometry detection system, comprising a matrix-assisted laser desorption ionization time-of-flight mass spectrometry system and the kit of the present application.

[0138] The present application adopts a nucleic acid mass spectrometry analysis system based on MALDI-TOF MS technology, and the time-of-flight mass spectrometry detection system (DP-TOF type, Di Spectrum Diagnosis) belongs to a high-precision DNA qualitative analysis platform. This technology platform perfectly integrates the high sensitivity of PCR technology, the high throughput of chip technology, the high accuracy of mass spectrometry technology, and the powerful function of computer intelligent analysis, providing a full-automatic solution with significant cost advantage, simple workflow, and high throughput for the market. The accuracy is ≥99.7%, and the salt removal, sample spotting, and detection are integrated and automated, which is simple, fast, and easy to operate, and the result interpretation is simple; 384 samples can be detected at one time, and the sample throughput is high, and a single sample can also be detected at one time, and the detection throughput is flexible; the site throughput is medium-high, and more than 40 sites can be detected in a single hole, which can reduce the use of precious samples, and the cost is as low as dozens of yuan, which saves the screening cost, reduces the national medical expenditure, is suitable for comprehensive promotion in different economic level regions in China, and is more suitable for large sample scientific research of related genes.

[0139] It should be understood that the various forms of the flow shown above can be reordered, added, or deleted steps. For example, the steps described in the present disclosure can be executed in parallel, in sequence, or in a different order, as long as the desired results of the technical solutions disclosed in the present disclosure can be achieved, which is not limited herein.

[0140] In addition, the terms "first", "second" are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "a plurality of" is two or more, unless otherwise specifically limited.

[0141] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed in the present disclosure, which should be covered within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the protection scope of the claims.

Claims

1. A reagent kit, characterized in that, The kit includes two sets of amplification primers, single-base extension primers, and processing reagents. The amplification primer set was used to amplify Mycobacterium tuberculosis flora identification sites, including IS6110, rd1, rd4, rd9, ext-rd9, rd12, and human internal reference gene sites IC2_1 and IC2_2; the amplification primer set was also used to amplify pharmacogene polymorphism sites of anti-tuberculosis drugs, including rrs905_2, pncA11, inhA15_2, gyrB499_2, rrs905_1, pncA76, katG315_2, rpoB516_2, rrs513_1, and gyrB4 99_1, rpsL43_1, rpoB516_1, gyrA90_1, inhA15_1, rpoB533, katG315_1, rpoB531, gyrA94_1, embB306_1, rpoB526_2, rpsL43_2, rpsL88_1, rpoB511, rrs513_2, gyrA94_2, embB306_2, rpoB526_1, rpsL88_2; each primer pair corresponds to the upstream and downstream regions of a SNP site. The two sets of amplification primers are the first set and the second set, respectively. The first set of amplification primers includes the detection sites: rrs905_2, rd4, IC2_1, ext-rd9, rd1, pncA11, inhA15_2, gyrB499_2, rrs905_1, pncA76, katG315_2, rpoB516_2, rrs513_1, gyrB499_1, rpsL43_1, rpoB516_1, gyrA90_1, inhA15_1, IS6110, rpoB533, rd12, katG315_1, and rpoB531; the amplification primer sequences for the detection sites included in the first set of amplification primers are shown in SEQ ID NO. 1~46 of the sequence listing; The second set of amplification primers includes the detection sites: gyrA94_1, IC2_2, embB306_1, rd9, rpoB526_2, rpsL43_2, rpsL88_1, rpoB511, rrs513_2, gyrA94_2, embB306_2, rpoB526_1, and rpsL88_2; the amplification primer sequences for the detection sites included in the second set of amplification primers are shown in SEQ ID NO. 47~72 of the sequence listing; The primer sequences of the single-base extension primers are shown in SEQ ID NO.73~108 of the sequence listing, respectively; The processing reagents include PCR reaction mixture, PCR enzyme mixture, SAP reaction mixture, SAP enzyme mixture, extension reaction mixture, and extension enzyme mixture.

2. The reagent kit according to claim 1, characterized in that, The sequences shown in SEQ ID NO. 96, SEQ ID NO. 103, and SEQ ID NO. 107 were designed with degenerate bases, and the 5' thio group was modified to prevent hydrolysis and the 3' MGB group was modified. The molecular weights of the thio group and the MGB group were 328.4 g / mol and 1120.6 g / mol, respectively.

3. The reagent kit according to claim 1, characterized in that, The processing reagent also includes an amplification reaction primer premix, which is a mixture of nucleotide sequences shown in SEQ ID NO. 1~72 of the sequence listing, with each amplification primer having a concentration of 0.3~3 μM.

4. The reagent kit according to claim 1, characterized in that, The processing reagent also includes a premixed solution of single-base extension reaction primers, which is a mixture of nucleotide sequences shown in SEQ ID NO. 73~108 of the sequence listing, with each single-base extension primer having a concentration of 5~30 μM.

5. The reagent kit according to claim 1, characterized in that, The kit also includes a desalination resin and a detection chip, the detection chip being a silicon-based chip containing 384 pre-dotted matrix detection points.

6. An integrated nucleic acid mass spectrometry detection system, characterized in that, Includes a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry system and a kit as described in any one of claims 1-5.

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