CN116064853A

CN116064853A - Kit and application thereof

Info

Publication number: CN116064853A
Application number: CN202210984317.8A
Authority: CN
Inventors: 康桃; 张盼; 潘军航; 余雯雯; 陈琦; 郭惠民; 张郁
Original assignee: Zhejiang Dipu Diagnosis Technology Co ltd
Current assignee: Zhejiang Dipu Diagnosis Technology Co ltd
Priority date: 2022-08-17
Filing date: 2022-08-17
Publication date: 2023-05-05

Abstract

The present disclosure provides a kit and applications thereof, the kit comprising two sets of amplification primer sets, a single base extension primer and a treatment reagent; the amplification primer group is used for amplifying mycobacterium tuberculosis flora identification sites and antituberculosis drug pharmaceutical gene polymorphism sites; each pair of primers corresponds to the upstream and downstream regions of one SNP locus respectively; the processing reagent comprises a PCR reaction mixed solution, a PCR enzyme mixed solution, an SAP reaction mixed solution, an SAP enzyme mixed solution, an extension reaction mixed solution and an extension enzyme mixed solution. By designing an amplification primer group and a single-base extension primer with excellent specificity, the sensitivity of an optimization system and a reagent is high, and the kit can complete detection of all sites by combining matrix-assisted laser desorption ionization time-of-flight mass spectrum, and has low detection cost and high efficiency.

Description

Kit and application thereof

Technical Field

The disclosure relates to the technical field of molecular diagnosis, in particular to a kit and application thereof.

Background

Tuberculosis is an infectious disease caused by mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), and according to the estimated world health organization, about 20 hundred million people worldwide are infected with the mycobacterium tuberculosis at present, about 300 ten thousand people die of tuberculosis every year, and especially the resistance of the mycobacterium tuberculosis to tuberculosis drugs is more remarkable in the epidemic and spread of tuberculosis in a large scale. The drug-resistant tubercle bacillus, especially Multi drug-resistant tubercle bacillus (MDR-TB) has fast transmission speed, rapid disease progress of patients and high death rate, and the death rate of some cases is only 4-6 weeks from diagnosis.

In general clinical laboratories, traditional methods such as absolute concentration, scale and resistance ratio methods are still used for the drug susceptibility detection of mycobacterium tuberculosis, and since mycobacterium tuberculosis grows slowly, it still takes 1-2 months to obtain results after isolated cultures are obtained, thus relying on traditional bacterial culture and drug susceptibility experiments can delay valuable treatment time. Although the recently developed BACTEC TM MGITTM 960 system can shorten the period of culturing tubercle bacillus and drug sensitivity experiment, it still takes about one week. Moreover, the instrumentation and reagents are quite expensive and therefore can only be developed in a very small number of hospitals. Numerous studies have shown that rapid nucleic acid detection of tubercle bacillus pathogens is more sensitive and more conducive to early clinical diagnosis than traditional sputum smears and tubercle bacillus culture assays.

At present, a medium flux technology based on matrix assisted laser desorption Ionization-Time of Flight (MALDI-TOF) is used as a medium flux technology in the field of gene detection, the detection range is wide, and the existing kit can only detect the gene polymorphism type of one gene/locus at a Time, and the specificity of an amplification primer is not high, so that a kit based on a matrix assisted laser desorption Ionization-Time of Flight (MALDI-TOF) platform for identifying mycobacterium tuberculosis flora and detecting drug-resistant mutation loci is needed.

Disclosure of Invention

The disclosure provides a kit and application thereof, which at least solve the problems of low specificity and low detection efficiency of amplification primers in the prior art.

According to a first aspect of the present disclosure, there is provided a kit comprising two sets of amplification primers, a single base extension primer, and a treatment reagent;

the amplification primer group IS used for amplifying mycobacterium tuberculosis flora identification sites, and comprises IS6110, rd1, rd4, rd9, ext-rd9 and rd12, and human internal reference gene sites IC2_1 and IC2_2; the amplification primer set is also used for amplifying anti-tubercular drug pharmaceutical gene 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, sL88_2; each pair of primers corresponds to the upstream and downstream regions of one SNP locus respectively;

the processing reagent comprises a PCR reaction mixed solution, a PCR enzyme mixed solution, an SAP reaction mixed solution, an SAP enzyme mixed solution, an extension reaction mixed solution and an extension enzyme mixed solution.

In one embodiment, the two sets of amplification primers are a first set of amplification primers and a second set of amplification primers, respectively, wherein,

the first amplification primer set includes a detection site: 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;

the second amplification primer set comprises 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.

In one embodiment, the first amplification primer set includes amplification primer sequences of detection sites respectively shown in sequence table SEQ ID NO. 1-46; the second amplification primer group comprises the amplification primer sequences of detection sites respectively shown in sequence table SEQ ID NO. 47-72.

In one embodiment, the primer sequences of the single-base extension primers are respectively shown in SEQ ID NO. 73-108 of the sequence table.

In one embodiment, the sequences shown in SEQ ID No.96, SEQ ID No.103, SEQ ID No.107 are degenerated and modified 5 'thio-hydrolysis modified, 3' MGB modified, thio and MGB having molecular weights of 328.4g/mol and 1120.6g/mol, respectively.

In one embodiment, the treatment reagent further comprises an amplification reaction primer premix, wherein the amplification reaction primer premix is a mixed solution of nucleotide sequences shown in SEQ ID NO. 1-72 of the sequence table, and the concentration of each amplification primer is 0.3-3 mu M.

In one embodiment, the treatment reagent further comprises a single-base extension primer premix, wherein the single-base extension primer premix is a mixed solution of nucleotide sequences shown in SEQ ID No. 73-108 of the sequence table, and the concentration of each single-base extension primer is 5-30 mu M.

In one embodiment, the kit further comprises a desalting resin and a detection chip comprising a silicon-based chip comprising 384 detection spots of the pre-spotted matrix.

According to a second aspect of the present disclosure there is provided the use of a kit for the identification of Mycobacterium tuberculosis flora and detection of drug-resistant mutation sites.

According to a third aspect of the present disclosure, 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 a kit according to the present invention.

According to the detection kit and the application thereof, the amplification primer group and the single-base extension primer with excellent specificity are designed, the optimized system and the reagent are high in sensitivity, detection of all sites can be completed by combining the kit with matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and the problems of high cost and low efficiency caused by repeated detection are avoided by acquiring all strain information and drug-resistant mutation site information of mycobacterium tuberculosis at one time with lower cost.

It should be understood that the description in this section is not intended to identify key or critical features of the embodiments of the disclosure, nor is it intended to be used to limit the scope of the disclosure. Other features of the present disclosure will become apparent from the following specification.

Drawings

The above, as well as additional purposes, features, and advantages of exemplary embodiments of the present disclosure will become readily apparent from the following detailed description when read in conjunction with the accompanying drawings. Several embodiments of the present disclosure are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings, in which:

in the drawings, the same or corresponding reference numerals indicate the same or corresponding parts.

1.1-1.4 in FIG. 1 is a sample No.1 detection result diagram by using the 1 st primer group, a DP-TOF nucleic acid mass spectrometer detects a software interface, and the software automatically judges the base type of each position according to the peak-to-peak condition of the product after extension of each hole;

2.1-2.4 in FIG. 2 is a sample No. 2 detection result diagram by using the 1 st primer group, a DP-TOF nucleic acid mass spectrometer detects a software interface, and the software automatically judges the base type of each position according to the peak-to-peak condition of the product after extension of each hole;

3.1-3.4 in FIG. 3 are graphs of the detection results of sample No.1 by the group 2 primers, the interface of the detection software of the DP-TOF nucleic acid mass spectrometer, and the software automatically judges the base type of each position according to the peak-to-peak condition of the product after the extension of each hole;

4.1-4.4 in FIG. 4 are graphs of the detection results of sample No. 2 by the group 2 primers, the interface of the detection software of the DP-TOF nucleic acid mass spectrometer, and the software automatically judges the base type of each position according to the peak-to-peak condition of the product after the extension of each hole;

FIG. 5 is a diagram of Sanger sequencing peaks for strain 1 (mutant in the gyrA 94-1 extension primer segment);

FIG. 6 is a diagram of mass spectrum detection peaks of strain 1 (mutant of the gyra94_1 extension primer segment);

FIG. 7 is a diagram of Sanger sequencing peaks for strain 2 (with mutations in the rpoB511 extension primer segment);

FIG. 8 is a mass spectrum detection peak diagram of strain 2 (mutant in rpoB511 extension primer segment);

FIG. 9 is a diagram of Sanger sequencing peaks for strain 3 (mutant rpoB526_1 extension primer segment);

FIG. 10 is a diagram of the mass spectrum detection peaks of strain 3 (mutant in rpoB526_1 extension primer segment).

Detailed Description

In order to make the objects, features and advantages of the present disclosure more comprehensible, the technical solutions in the embodiments of the present disclosure will be clearly described in conjunction with the accompanying drawings in the embodiments of the present disclosure, and it is apparent that the described embodiments are only some embodiments of the present disclosure, but not all embodiments. Based on the embodiments in this disclosure, all other embodiments that a person skilled in the art would obtain without making any inventive effort are within the scope of protection of this disclosure.

Example 1

The invention provides a kit, which comprises two amplification primer groups, a single-base extension primer and a treatment reagent;

the amplification primer set IS used for amplifying mycobacterium tuberculosis flora identification sites, including IS6110, rd1, rd4, rd9, ext-rd9 and rd12, human internal reference gene loci IC2_1 and IC2_2, and IS also used for amplifying antitubercular pharmaceutical gene 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, rpL43_2, sL88_1, rpoB511, rps2_grWrW1, rpSb2_4rW1, rpL2_4rW1; each pair of primers corresponds to the upstream and downstream regions of one SNP locus respectively;

The two amplification primer sets are respectively a first amplification primer set and a second amplification primer set, wherein,

The amplification primers and single base extension primers were synthesized by Shanghai Bai Ge Biotechnology Co., ltd, and the sequences are shown in Table 1 and Table 2.

TABLE 1 amplification primer sequences

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TABLE 2 Single base extension primer sequences

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In one example, the sequences shown in sequence listings SEQ ID No.96, SEQ ID No.103, SEQ ID No.107 were degenerated, 5 'thio-hydrolysis modified, 3' MGB modified, thio and MGB molecular weights of 328.4g/mol and 1120.6g/mol, respectively.

In one example, the processing reagent includes a PCR reaction mixture, a PCR enzyme mixture, an SAP reaction mixture, an SAP enzyme mixture, an extension reaction mixture, an extension enzyme mixture.

The PCR reaction mixture comprises: PCR buffer, mg ²⁺ dATP, dCTP, dTTP, dUTP and dGTP, etc.;

the PCR enzyme mixture comprises: taq enzyme and UNG enzyme;

the SAP reaction mixture comprises: SAP buffer;

the SAP enzyme mixed solution comprises the following components: SAP enzyme and enzyme preservation solution;

the extension reaction mixture comprises: extension buffer, mg ²⁺ ddATP, ddCTP, ddTTP, ddGTP, etc

The elongase mixed solution comprises: elongase, enzyme preservation solution, and glycerol.

In one example, the processing reagent further comprises an amplification reaction primer pre-mix, which is a mixture of nucleotide sequences shown in sequence listing SEQ ID NO. 1-72, at a concentration of 0.3-3. Mu.M for each amplification primer.

In one example, the treatment reagent further comprises a single base extension reaction primer pre-mix of nucleotide sequences shown in sequence listing SEQ ID NOS.73-108, each single base extension primer having a concentration of 5-30. Mu.M.

In one example, the kit further comprises:

desalting resin: comprises cation exchange resin powder for removing salt ions of an extension reaction solution;

and (3) a detection chip: comprising a silicon-based chip containing 384 pre-spotted substrates;

pure mutant control: the concentration of the plasmid aqueous solution containing 21-site mutant mycobacterium tuberculosis drug-resistant gene corresponding fragment diluted after absolute quantification by a fluorescence quantitative standard curve method is more than 1000 copies/. Mu.l;

hybrid control: the method comprises the steps of absolute quantification of a diluted 21-site mutant type and wild mycobacterium tuberculosis drug-resistant gene corresponding fragment plasmid mixed aqueous solution by a fluorescence quantitative standard curve method, wherein the concentration is more than 1000 copies/mu l;

pure wild control: the concentration of the plasmid aqueous solution containing 21 wild mycobacterium tuberculosis drug-resistant genes corresponding fragments diluted after absolute quantification by a fluorescence quantification standard curve method is more than 1000 copies/. Mu.l.

The kit provided by the invention is used for identifying mycobacterium tuberculosis flora and detecting drug-resistant mutation sites, an amplification primer group and a single-base extension primer with excellent specificity are designed, an optimized system and reagent sensitivity are high, detection of all sites can be completed by combining the kit with matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and the problems of high cost and low efficiency caused by repeated detection are avoided.

Example 2

An application method of a kit for identifying mycobacterium tuberculosis flora and detecting drug-resistant mutation sites, wherein the kit is provided by the invention; the method comprises the following steps:

s1, preparing a mycobacterium tuberculosis DNA template;

firstly, collecting a sample, wherein the sample can be sputum, alveolar lavage fluid, bacterial culture fluid, cotton swab, body fluid, excrement and the like of a patient. Next, bacterial genomic DNA was extracted from the obtained sample by the procedure of extracting the instruction sheet from the Ex-DNA bacterial genome (proprietary, cat. T132) of Tianlong technology in this example.

S2, detecting the extracted bacterial genome DNA according to the 2 groups of amplification primer groups and the 2 groups of single-base extension primer groups;

the step S2 comprises the following steps:

s21, preparing a PCR reagent;

the PCR reaction mixture and the PCR enzyme mixture were taken out from the kit, melted and mixed at room temperature, and centrifuged at 2000rpm for 10sec, respectively, to calculate the number of people to be prepared with the reagents.

The PCR test reaction system comprises: PCR reaction mix 1.67. Mu. L, PCR enzyme mix 0.33. Mu. L, PCR amplification primer premix 1. Mu.L.

According to the PCR test reaction system, the use amount of each reagent is calculated, fully and uniformly mixed, and then the mixture is split into a PCR reaction tube or a 384-well PCR plate according to the amount of 3 mu L and transferred to a sample processing area.

S22, loading a sample;

mu.L of the bacterial genomic DNA solution extracted in step S1 was added, respectively, and the reaction tube or 384-well PCR plate was capped and then transferred to the detection zone.

S23, PCR amplification reaction;

placing each reaction tube on a PCR instrument according to a certain sequence, and carrying out PCR amplification according to the following procedures:

the PCR product was temporarily not subjected to the next experimental procedure and was kept at 4℃overnight.

Under the guidance of the primer, the DNA template (namely the extracted bacterial genome DNA) is subjected to the steps of pre-denaturation, extension and annealing in sequence to complete the PCR amplification reaction.

S24, preparing SAP reaction liquid;

the SAP reaction mixture and the SAP enzyme mixture were removed from the kit, melted and mixed with shaking at room temperature, and centrifuged at 2000rpm for 10sec. The number of parts of the reagent to be prepared is calculated.

Each test reaction system included: SAP reaction mixture 1.70 mu L, SAP enzyme mixture 0.30 mu L.

S25, sample adding (nucleic acid amplification region);

to the PCR amplification product of S23, 2. Mu.L of the above SAP reaction solution was added, and the reaction tube or 384-well PCR plate was capped.

The reaction tubes were placed in a PCR apparatus in the order given, SAP digestion was performed according to the following procedure:

the SAP product should be immediately subjected to the next step, and it is not recommended to leave it at 4℃overnight.

S26, preparing an extension reagent;

the extension reaction mixture, the elongase mixture and the single-base extension reaction premix were removed from the kit, melted at room temperature, mixed by shaking, and centrifuged at 2000rpm for 10sec. The number of parts of the reagent to be prepared is calculated.

Each test reaction system included: extension reaction mixture 0.72. Mu.L, extension enzyme mixture 0.34. Mu.L, single base extension reaction premix 0.94. Mu.L.

S27, sample adding (nucleic acid amplification region);

to the SAP product of S26, 2. Mu.L of the above extension reaction solution was added, respectively, and the reaction tube or 384-well PCR plate was capped.

Placing each reaction tube on a PCR instrument according to a certain sequence, and performing extension amplification according to the following procedures:

if the extension product is not subjected to the next experimental procedure, it can be stored overnight at 4 ℃.

S28, performing mass spectrum detection (amplification analysis region) by using a DP-TOF time-of-flight mass spectrum detection system:

standard operation is carried out according to the operation instruction of the DP-TOF nucleic acid mass spectrometer, the chip and the hole number corresponding to the required detection sample are selected, the instrument automatically carries out desalting, chip sample application and detection on the sample extension product, and the result is automatically analyzed.

S29, importing the result file analyzed by the mass spectrometer into a sample result reporting system, and issuing a sample result report.

For example, FIGS. 1 to 4 show graphs of the detection results of sample No.1 and sample No. 2 using the first primer set and the second primer set, respectively. Taking fig. 1 as an example, fig. 1.1 is a schematic diagram for explaining whether the detection result of the first set of primers is good or not for the sample 1, wherein the circle "≡o" is an unknown sample, and the diamond "facto" is a blank control; the dark green color represents the best detection result, the light green color, the yellow color again and the worst red color result, and because the mass spectrum detection result page is colored and the drawing of the application is black and white, the drawing of the application distinguishes the dark green, the light green, the yellow and the red of the mass spectrum software interface by the color shade, wherein the red is the darkest color, the dark green color, the yellow color again and the light green is the lightest. There is no yellow color in fig. 1.1 of the present application, for example, in fig. 1.1, M rows 18 columns and 20 columns, P rows 20 columns and 24 columns are red; m rows 22 columns, N rows 19 columns, O rows 20 columns and 23 columns, P rows 18 columns are light green, and the rest are dark green. FIGS. 1.2 and 1.3 show the peak-to-peak condition of the product after extension of each well, and FIG. 1.4 shows the automatic interpretation of the base type at each position by the system.

Example 3

The kit is used for detecting the accuracy of the drug-resistant mutation site of the mycobacterium tuberculosis, and the method comprises the following steps:

(1) Extracting 10 cases of clinical tuberculosis sputum samples known to be positive for the mycobacterium tuberculosis, and extracting tuberculosis genome DNA;

(2) 10 tuberculosis sputum samples were tested using 2 sets of primers according to example 2;

(3) The detection accuracy of the kit is verified by adopting a Sanger sequencing method with a sequence gold standard, and verification primer pairs for amplifying the areas where all the sites are located are synthesized by Shanghai Bai Ge biotechnology limited company, and the sequences are shown in table 3.

TABLE 3 corresponding site sequences

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

(5) The reaction mixture was mixed and centrifuged at 8000g for rapid centrifugation, and the PCR was verified as follows:

(6) After PCR amplification of each sample was completed, the amplified fragments were determined by 2% agarose electrophoresis and were sent to sequencing company (Hangzhou Shangya Biotechnology Co., ltd.) for sequencing verification, and the results were compared with the mass spectrometry detection results. Taking an example of the results, the comparison results are shown in table 4 below.

Table 4 results of comparison

Conclusion: sanger sequencing detection results were completely consistent with nucleic acid flight mass spectrometry results.

The invention has the advantages that: the 2-well reaction detects all drug-resistant mutation sites, while the traditional sequencing method requires 13-well reaction, and the workload and the cost are far higher than those of the method.

Example 4

The kit provided by the invention is used for identifying mycobacterium tuberculosis flora and detecting sensitivity of drug-resistant mutation sites.

In this example, 2 samples of DNA extracted from national reference of Mycobacterium tuberculosis in China were selected, subjected to gradient dilution, the Qubit spectrophotometer determines the initial DNA concentration, the initial DNA loading amounts are respectively 0.5 ng/. Mu.l, 0.1 ng/. Mu.l, 0.05 ng/. Mu.l, 0.025 ng/. Mu.l and 0.001 ng/. Mu.l, the subsequent PCR, digestion, extension and mass spectrometry are carried out, and the statistics of the 2 samples are shown in tables 5 to 6 below.

TABLE 5 sample 1 sensitivity test results

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TABLE 6 sample 2 sensitivity test results

According to the test results shown in tables 5 and 6, all sites of 2 samples were correctly detected when the loading amount was as low as 0.05 ng/. Mu.l, and only individual sites were detected when the loading amount was 0.025 ng/. Mu.l. It is demonstrated that the kit can detect all sites at one time with very high sensitivity under the condition of low to 0.05 ng/. Mu.l of bacterial genome DNA.

Example 5

The kit can detect the variant type of the strain with the base mutation in the extension primer segment through degenerate primer design.

In this example, 3 Mycobacterium tuberculosis strains were selected, whose extension primer segment was confirmed to have a base mutation by Sanger sequencing, strain 1 (mutation of the extension primer segment of gyrA 94-1), strain 2 (mutation of the extension primer segment of rpoB 511), and strain 3 (mutation of the extension primer segment of rpoB526-1); the results of the assays performed using the kits of the invention are shown, for example, in FIGS. 5-10.

In the kit, the primers SEQ ID NO.96, SEQ ID NO.103 and SEQ ID NO.107 are designed with degenerate bases, and the result shows that the kit can also detect corresponding sites for strains with base mutation in the extended primer segment, and is suitable for researching complex samples or rare samples.

In yet another aspect, the invention provides an integrated nucleic acid mass spectrometry detection system comprising a matrix-assisted laser desorption ionization time-of-flight mass spectrometry system and a kit according to the invention.

The invention adopts a nucleic acid mass spectrometry system based on MALDI-TOF MS technology, and a time-of-flight mass spectrometry detection system (DP-TOF, di-spectrum diagnosis) belongs to a high-precision DNA qualitative analysis platform. The technical platform perfectly integrates the high sensitivity of the PCR technology, the high flux of the chip technology, the high accuracy of the mass spectrum technology and the powerful function of computer intelligent analysis, and provides a full-automatic solution with obvious cost advantages, simple workflow and high flux for the market. The accuracy is more than or equal to 99.7%, the desalting, sample application and detection are integrated, the automation degree is high, the operation is simple and quick, and the result interpretation is simple; 384 samples can be detected at a time, the flux of the detected samples is high, a single sample can be detected at a time, the detection flux is flexible, and the detection can be carried out along with the detection; the site flux is high, and the single hole can detect more than 40 sites at most, so that the consumption of precious samples can be reduced, the cost is as low as tens of yuan, the screening cost is saved, the national medical expenditure is reduced, the method is suitable for comprehensive popularization in different economic level areas of the whole country, and the method is more suitable for large sample scientific research application of related genes.

It should be appreciated that various forms of the flows shown above may be used to reorder, add, or delete steps. For example, the steps described in the present disclosure may be performed in parallel, sequentially, or in a different order, provided that the desired results of the technical solutions of the present disclosure are achieved, and are not limited herein.

Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present disclosure, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.

The foregoing is merely specific embodiments of the disclosure, but the protection scope of the disclosure is not limited thereto, and any person skilled in the art can easily think about changes or substitutions within the technical scope of the disclosure, and it is intended to cover the scope of the disclosure. Therefore, the protection scope of the present disclosure shall be subject to the protection scope of the claims.

Claims

1. A kit, characterized in that the kit comprises two amplification primer sets, a single base extension primer and a treatment reagent;

2. The kit of claim 1, wherein the two sets of amplification primers are a first set of amplification primers and a second set of amplification primers, respectively, wherein,

3. The kit according to claim 2, wherein the first amplification primer set comprises amplification primer sequences of detection sites shown in sequence table SEQ ID NO. 1-46, respectively; the second amplification primer group comprises the amplification primer sequences of detection sites respectively shown in sequence table SEQ ID NO. 47-72.

4. The kit according to claim 2, wherein the primer sequences of the single-base extension primers are shown in sequence tables SEQ ID NO. 73-108, respectively.

5. The kit according to claim 4, wherein the sequences shown in SEQ ID No.96, SEQ ID No.103 and SEQ ID No.107 are subjected to degenerate base design, 5 'thio hydrolysis modification, 3' MGB modification, thio and MGB molecular weights of 328.4g/mol and 1120.6g/mol, respectively.

6. The kit according to claim 3, wherein the treatment reagent further comprises an amplification reaction primer pre-mixture, wherein the amplification reaction primer pre-mixture is a mixture of nucleotide sequences shown in SEQ ID NOS.1 to 72 in the sequence Listing, and each amplification primer has a concentration of 0.3 to 3. Mu.M.

7. The kit according to claim 4, wherein the treatment reagent further comprises a single-base extension primer premix, the single-base extension primer premix being a mixture of nucleotide sequences represented by SEQ ID NOS.73 to 108 in the sequence Listing, and each single-base extension primer having a concentration of 5 to 30. Mu.M.

8. The kit of claim 1, further comprising a desalting resin and a detection chip comprising a silicon-based chip comprising 384 detection spots of pre-spotted matrix.

9. Use of a kit according to any one of claims 1 to 8, wherein the kit is for the identification of mycobacterium tuberculosis flora and detection of drug-resistant mutation sites.

10. 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 any one of claims 1-8.