Primer group for detecting drug resistance of mycobacterium tuberculosis and rifampicin, kit and application

Abstract

The invention relates to a primer group for detecting drug resistance of mycobacterium tuberculosis and rifampicin, a kit and application. The primer group disclosed by the invention comprises multiple PCR (Polymerase Chain Reaction) primers aiming at a mycobacterium tuberculosis IS6110 gene, an IS1081 gene and an rpoB gene, and the rpoB gene region comprises three amplification forward primer sequences corresponding to a 516 codon region, a 526 codon region and a 531 codon region, two locking sequences (LNA) corresponding to the amplification forward primer sequences and one amplification reverse primer sequence corresponding to the amplification forward primer sequences. The primer group can be used for quickly and accurately identifying the mycobacterium tuberculosis and identifying the rifampicin resistance of the mycobacterium tuberculosis; a result measured by the method is good in specificity and high in sensitivity, and can be directly read by naked eyes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer set, a kit and applications for detecting Mycobacterium tuberculosis and rifampicin resistance. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB). The high prevalence and transmissibility of drug-resistant TB pose a significant threat to public health. Early diagnosis and timely treatment are key to effectively controlling the spread of TB and restoring the health of TB patients.

[0003] In recent years, with the rapid development of molecular biological diagnostic technology, it has provided a key approach for the rapid diagnosis of tuberculosis and drug-resistant tuberculosis. These detection methods based on molecular biological principles omit the tedious process of traditional bacterial culture, thereby significantly reducing the time required for the diagnosis of tuberculosis and its drug-resistant types. IS6110 is an insertion sequence of Mycobacterium tuberculosis and is also the most commonly used conserved sequence in the molecular diagnosis of tuberculosis. However, IS6110 is still missing in 10% of strains, affecting the accuracy of detection. IS1081 is another common insertion sequence in the Mycobacterium tuberculosis group. It has a low copy number in the strain, but is stably present in all MTB. The combined detection of the two insertion sequences IS6110 and IS1081 can avoid the problem of false positive detection and improve the accuracy of detection.

[0004] With the deepening of research, the problem of heterogeneous rifampicin resistance in Mycobacterium tuberculosis has become increasingly prominent in clinical practice. Rapid and accurate resistance testing is crucial for early detection of the disease and selection of sensitive drugs. Traditional drug susceptibility testing is time-consuming (2-3 months) and cannot meet clinical needs. Rifampicin resistance is closely associated with mutations in the rpoB gene of Mycobacterium tuberculosis. 95% of mutations are concentrated within the 81-base resistance-determining region, and mutations at codons 516, 526, and 531 are responsible for over 90% of drug-resistant strains.

[0005] Based on this, this invention was proposed. This invention can jointly detect the two insertion sequences of IS6110 and IS1081 to improve the accuracy of molecular detection; at the same time, it can detect the high mutation sites in the rpoB region and determine the drug resistance of the strain. Summary of the Invention

[0006] The present invention aims to provide a primer set for detecting Mycobacterium tuberculosis and rifampicin resistance. This primer set is designed to jointly detect the IS6110 and IS1081 insertion sequences in Mycobacterium tuberculosis and simultaneously detect mutations at codons 516, 526, and 531 of the rpoB gene. This method can simultaneously diagnose Mycobacterium tuberculosis and determine its rifampicin resistance.

[0007] The present invention specifically adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a primer set for detecting Mycobacterium tuberculosis and rifampicin resistance, the primer set comprising a multiplex PCR primer set, an amplification primer targeting the drug resistance site of the rpoB gene of Mycobacterium tuberculosis, and a locked nucleic acid probe;

[0009] The multiplex PCR primer set includes IS6110_F, IS6110_R, IS1081_F and IS1081_R primers;

[0010] The sequence of the IS6110_F primer is shown in SEQ ID NO.1;

[0011] The sequence of the IS6110_R primer is shown in SEQ ID NO.2;

[0012] The sequence of the IS1081_F primer is shown in SEQ ID NO.3;

[0013] The sequence of the IS1081_R primer is shown in SEQ ID NO.4;

[0014] The amplification primers and locked nucleic acid probes targeting the drug resistance site of the Mycobacterium tuberculosis rpoB gene include:

[0015] The forward rpoB-F1 primer sequence targeting codon 516 of the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO.5.

[0016] The forward rpoB-F2 primer sequence targeting codon 526 of the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO.6.

[0017] The forward rpoB-F3 primer sequence targeting codon 531 of the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO.7.

[0018] The locked LNA-1 primer sequence targeting codon 516 of the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO.8.

[0019] The locked LNA-2 primer sequence targeting codons 526 and 531 of the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO.9.

[0020] The reverse rpoB-R primer sequence targeting the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO: 10.

[0021] Furthermore, it also includes A1, A2, L1, L2, L3, ST-DNA and ST-cDNA primers for PCR product recognition and signal output;

[0022] The sequence of the A1 primer is shown in SEQ ID NO.11;

[0023] The sequence of the A2 primer is shown in SEQ ID NO.12;

[0024] The sequence of the L1 primer is shown in SEQ ID NO.13;

[0025] The sequence of the L2 primer is shown in SEQ ID NO.14;

[0026] The sequence of the L3 primer is shown in SEQ ID NO.15;

[0027] The sequence of the ST-DNA primer is shown in SEQ ID NO.16;

[0028] The sequence of the ST-cDNA primer is shown in SEQ ID NO.17.

[0029] Furthermore, the 3' end of the sequence of SEQ ID NO. 8 is modified with an NH2-C6 group, the 3' end of the sequence of SEQ ID NO. 9 is modified with an NH2-C6 group, and the 5' end of the sequence of SEQ ID NO. 16 is modified with an SH group.

[0030] In a second aspect, the present invention provides a kit for detecting Mycobacterium tuberculosis and rifampicin resistance, comprising the primer set for detecting Mycobacterium tuberculosis and rifampicin resistance.

[0031] Furthermore, it includes 2 volumes of 1 μM IS6110_F primer, IS6110_R primer, IS1081_F primer, IS1081_R primer, rpoB-F1 primer, rpoB-F2 primer, rpoB-F3 primer, rpoB-R primer, LNA-1 primer, LNA-2 primer, 5 volumes of PCR buffer, 3 volumes of 25 mM MgCl2, 1 volume of 10 mM dNTP, 1 volume of 5 U / μL Taq DNA polymerase and 17 volumes of ddH2O.

[0032] In a third aspect, the present invention provides a Mycobacterium tuberculosis and rifampicin resistance detection system, comprising a genome sequence extraction module, a PCR amplification module, a mixing reaction module, a Mycobacterium tuberculosis determination module, and a rifampicin resistance determination module;

[0033] The genome sequence extraction module is used to extract the genome sequence of the sample to be tested;

[0034] The PCR amplification module is used to perform multiplex PCR amplification on the sample to be tested using the primer set or the kit;

[0035] The mixed reaction module is used to denature the amplified product and perform a mixed reaction with the DNA "gate" structure composed of A1, A2 and L1, L2, L3 primers;

[0036] The Mycobacterium tuberculosis determination module is used to mix the mixed reaction product with hemin, react with TMB color developing solution, and determine Mycobacterium tuberculosis based on color changes;

[0037] The rifampicin resistance determination module is used to mix the mixed reaction product with the dsDNA-AuNPs solution incubated with ST-DNA and ST-cDNA and react, add NaCl solution after the reaction, and judge the rifampicin resistance of Mycobacterium tuberculosis based on the color change.

[0038] Furthermore, the reaction procedure of the multiplex PCR amplification includes 95°C for 5 min; 95°C for 30 s, 55°C for 35 s, 72°C for 1 min, 25 cycles; and 72°C for 10 min.

[0039] Furthermore, the reaction temperature of the mixed reaction is 37°C, and the reaction time is 30 min; the hemin concentration is 3 μM, the reaction temperature with the color developing solution is room temperature, the time is 10 min, and the amount of color developing solution added is 100 μL; the reaction temperature of the dsDNA-AuNPs solution incubated with ST-DNA and ST-cDNA and then mixed is 37°C, the reaction time is 30 min, the concentration of the NaCl solution is 0.2 M, and the volume is 100 μL.

[0040] In a fourth aspect, the present invention provides a method for detecting Mycobacterium tuberculosis and rifampicin resistance, comprising:

[0041] Extracting the genome sequence of the sample to be tested;

[0042] Performing multiplex PCR amplification on the sample to be tested using the primer set or the kit;

[0043] The amplified product is denatured and mixed with a DNA "gate" structure consisting of primers A1, A2 and L1, L2, and L3;

[0044] The mixed reaction product is mixed with hemin, reacted with TMB color developing solution, and Mycobacterium tuberculosis is identified based on the color change;

[0045] The mixed reaction product was mixed with the dsDNA-AuNPs solution incubated with ST-DNA and ST-cDNA and reacted. After the reaction, NaCl solution was added, and the rifampicin resistance of Mycobacterium tuberculosis was judged based on the color change.

[0046] In a fifth aspect, the present invention provides use of the primer set or the kit in preparing a reagent for detecting rifampicin-resistant Mycobacterium tuberculosis.

[0047] The beneficial effects of the present invention are:

[0048] (1) The combined detection of the two insertion sequences of IS6110 and IS1081 avoids the false negatives caused by the absence of a single insertion sequence in some strains and improves the accuracy of the detection.

[0049] (2) At the same time, the rpoB gene region of Mycobacterium tuberculosis was tested. Mutations in codons 516, 526, and 531 resulted in the production of more than 90% of drug-resistant strains. Testing these four sites can identify the vast majority of rifampicin-resistant strains.

[0050] (3) The detection of Mycobacterium tuberculosis and its drug resistance detection can be performed simultaneously, which is convenient to operate and saves detection time.

[0051] (4) The output signals are different colorimetric signals, and the test results can be read directly by the naked eye, which is suitable for tuberculosis detection in underdeveloped areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0053] Figure 1 Schematic diagram of the detection of Mycobacterium tuberculosis and its rifampicin resistance.

[0054] Figure 2 Agarose gel electrophoresis verification of different fragments in multiplex PCR.

[0055] Figure 3This is the optimized diagram of the number of base pairings at each position of the DNA "gate" structure; Figure 3 A is the probability of combining two sequences with different numbers of bases, Figure 3 B is the Tm value of the two sequences with different base numbers, Figure 3 C is the probability of combining the three sequences with different numbers of bases.

[0056] Figure 4 This is the polyacrylamide electrophoresis verification diagram of the synthesized DNA "gate" structure.

[0057] Figure 5 The graph shows the absorbance change before and after TMB color development.

[0058] Figure 6 This is a curve of absorbance change before and after gold nanoparticle aggregation. DETAILED DESCRIPTION

[0059] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0060] Example 1 Design of sequence groups

[0061] The primer-probe combination for detecting rifampicin resistance of Mycobacterium tuberculosis of the present invention is shown in Table 1:

[0062] Table 1 Primer-probe combinations for detection of rifampicin resistance in Mycobacterium tuberculosis

[0063]

[0064]

[0065] Example 2 PCR reaction system

[0066] The PCR reaction system for detecting Mycobacterium tuberculosis and its rifampicin resistance of the present invention is shown in Table 2.

[0067] Table 2 PCR reaction system for detection of Mycobacterium tuberculosis and its rifampicin resistance

[0068]

[0069]

[0070] Example 3 Mycobacterium tuberculosis and rifampicin resistance detection process

[0071] Extract the genome sequence of the sample to be tested.

[0072] The above primer set was used to perform multiplex PCR amplification on the test samples; wherein, the concentration of each primer in the primer set was 1 μM, the MgCl2 concentration was 25 mM, the dNTP concentration was 10 mM, and the Taq DNA polymerase was 5 U / μL; the reaction procedure for the multiplex PCR amplification included 95°C for 5 min; 95°C for 30 s, 55°C for 35 s, and 72°C for 1 min, 25 cycles; and 72°C for 10 min.

[0073] The amplified product was placed at 95°C for 5 min for denaturation and then immediately placed in an ice box to maintain the denatured state.

[0074] The denatured amplified product was mixed with a DNA "gate" structure composed of primers A1, A2 and L1, L2, and L3; the reaction was carried out at 37°C for 30 minutes.

[0075] The mixed reaction product was mixed with hemin, reacted with TMB colorimetric solution, and Mycobacterium tuberculosis was identified based on the color change; the hemin concentration was 3 μM, the reaction was carried out at 37°C for 30 minutes; the volume of colorimetric solution added was 100 μL, and the color development reaction was carried out at room temperature for 10 minutes.

[0076] The mixed reaction product was mixed with the dsDNA-AuNPs solution incubated with ST-DNA and ST-cDNA and reacted. After the reaction, NaCl solution was added, and the rifampicin resistance of Mycobacterium tuberculosis was judged based on the color change. After mixing, the reaction was carried out at 37°C for 30 minutes. The concentration of the added NaCl solution was 0.2M and the volume was 100 μL. The color development reaction was carried out at room temperature for 5 minutes.

[0077] The incubation process is as follows: 25 μL of 2 μM ST-DNA is mixed with 10 μL of 500 μM TCEP and incubated at room temperature for 1 hour to reduce and activate the sulfhydryl groups; 25 μL of 2 μM ST-cDNA and 40 μL of gold nanoparticle solution are added to form dsDNA-AuNPs.

[0078] Example 4 Primer set for detection of Mycobacterium tuberculosis and rifampicin resistance and its application, the specific principle is:

[0079] See attached Figure 1, two auxiliary primers A and three locking primers L can be combined to form a DNA "OR gate" structure. The sample genome is subjected to multiple PCR amplification, where L1 can bind to the IS1081 fragment amplification product, and L2 can bind to the IS6110 fragment amplification product. Due to the design of the binding energy, any binding of L1 and L2 primers to the target amplification fragment will cause A1 to fall off. A fragment of DNA nanozyme is designed in the sequence of primer A1, which can form a G quadruplex and trigger the subsequent TMB color development reaction to achieve the detection of Mycobacterium tuberculosis.

[0080] Mutations at sites 516, 526, and 531 in the rpoB gene region confer rifampicin resistance in 90% of strains. Locked nucleic acid probes were designed targeting these three sites. When none of the three sites are mutated, no PCR product targeting the rpoB fragment is generated, and L2 binds tightly to L2, preventing further reactions. However, mutations at any of these sites allow PCR amplification of the rpoB region. The amplified product binds to L3, releasing A2, causing subsequent aggregation and discoloration of gold nanoparticles, thus enabling detection of rifampicin resistance.

[0081] Example 5 Agarose gel electrophoresis verification of different fragments in multiplex PCR

[0082] In order to evaluate the amplification specificity of each target sequence in the multiplex PCR system, a 2% agarose gel was used to prepare a horizontal electrophoresis platform. The selection of 2% gel concentration was based on the optimization results of the matching between the target product fragment size (IS6110: 317bp, IS1081: 111bp, rpoB: 247bp) and the gel pore size. The electrophoresis buffer used 1×TBE Buffer as the buffer, and 10μL PCR product and 2μL 6×Loading Buffer were loaded. In order to obtain clear resolution, the constant voltage was set to 110V, and the electrophoresis time was controlled at 35-40 minutes. Figure 2 As shown, the products of the designed IS6110, IS1081 and rpoB regions were successfully amplified, with obvious bands and no mixed bands.

[0083] Example 6 Optimization of the number of base pairings at each binding position of the DNA "gate" structure

[0084] In view of the dynamic assembly characteristics of DNA "AND gate" structure, NUPACK 3.4.0 software was used to systematically optimize the base pairing of binding sites. Through the discretization scanning strategy, the effect of the number of bases in different binding sites on the binding stability was investigated. Figure 3 As shown in Figures A and 3B, as the number of paired bases at each site decreases, the Gibbs free energy of the DNA connection decreases, resulting in a downward trend in the Tm value and binding probability of the primer. Figure 3C analyzed the binding probabilities for different pairing scenarios. To ensure successful assembly of the DNA "gate" structure and that any L primer would release the A1 primer upon binding to the target, the number of bases in the circle was selected as the final number of bases used for L1 and L2. Because the L3 primer functionally requires a strong binding to A2, the L3-A2 junction remained at 12 bases, without any base reduction.

[0085] Example 7: Polyacrylamide electrophoresis verification of DNA “gate” structure after synthesis

[0086] To verify the correct assembly of the "OR gate" DNA nanostructure, a non-denaturing vertical electrophoresis system was used for characterization. A 12% polyacrylamide gel was prepared with a thickness of 1.0 mm and a sample well volume of 15 μL. The electrophoresis buffer was 1×TBE Buffer. 10 μL of the assembly product was mixed with 2 μL of 6×Loading Buffer, and 10 μL was loaded. The constant voltage was set to 115 V, and electrophoresis was performed for 50 minutes until the bromophenol blue indicator migrated to the bottom of the gel. The gel was then removed for staining. Figure 4 As shown, the fully assembled "OR gate" structure presents a single migration band (lane 9), which is significantly lower than the monomers of each component, indicating that the DNA structure was successfully synthesized.

[0087] Feasibility Verification of the Method in Example 8 for Detection of Mycobacterium tuberculosis

[0088] The reaction product was developed with TMB and its absorbance was scanned. Figure 5 As can be seen, when the target IS6110 and IS1081 fragments in the system are amplified, a significant absorbance change is observed, indicating that the target is detected; when the target is not present in the system, neither genetic fragment can be amplified, the subsequent colorimetric reaction cannot be triggered, and there is no significant absorbance change. This method can effectively detect Mycobacterium tuberculosis.

[0089] Feasibility Verification of the Method in Example 9 for Detecting Rifampicin Resistance in Mycobacterium tuberculosis

[0090] The reaction product was mixed with the dsDNA-AuNPs solution and its absorbance was scanned after the color change, such as Figure 6 As shown, when the concentration of amplification primers was 1 μM, the color of the system changed significantly. As the concentration decreased, the color change of the gold nanoparticles became less obvious, indicating that it can effectively distinguish rifampicin resistance of Mycobacterium tuberculosis.

[0091] The detection performance evaluation of the method in Example 10 is shown in Table 3.

[0092] Table 3 Detection performance of the proposed method evaluated on clinical samples

[0093]

[0094] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A primer set for detecting Mycobacterium tuberculosis and rifampicin resistance, characterized in that: The primer set includes a multiplex PCR primer set, an amplification primer targeting the drug resistance site of the rpoB gene of Mycobacterium tuberculosis, and a locked nucleic acid probe; The multiplex PCR primer set includes IS6110_F, IS6110_R, IS1081_F and IS1081_R primers; The sequence of the IS6110_F primer is shown in SEQ ID NO.1; The sequence of the IS6110_R primer is shown in SEQ ID NO.2; The sequence of the IS1081_F primer is shown in SEQ ID NO.3; The sequence of the IS1081_R primer is shown in SEQ ID NO.4; The amplification primers and locked nucleic acid probes targeting the drug resistance site of the Mycobacterium tuberculosis rpoB gene include: The forward rpoB-F1 primer sequence targeting codon 516 of the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO.

5. The forward rpoB-F2 primer sequence targeting codon 526 of the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO.

6. The forward rpoB-F3 primer sequence targeting codon 531 of the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO.

7. The locked LNA-1 primer sequence targeting codon 516 of the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO.

8. The locked LNA-2 primer sequence targeting codons 526 and 531 of the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO.

9. The reverse rpoB-R primer sequence targeting the rpoB gene of Mycobacterium tuberculosis is shown in SEQ ID NO:

10.

2. The primer set for detecting Mycobacterium tuberculosis and rifampicin resistance according to claim 1, characterized in that: Also included are A1, A2, L1, L2, L3, ST-DNA, and ST-cDNA primers for PCR product recognition and signal output; The sequence of the A1 primer is shown in SEQ ID NO.11; The sequence of the A2 primer is shown in SEQ ID NO.12; The sequence of the L1 primer is shown in SEQ ID NO.13; The sequence of the L2 primer is shown in SEQ ID NO.14; The sequence of the L3 primer is shown in SEQ ID NO.15; The sequence of the ST-DNA primer is shown in SEQ ID NO.16; The sequence of the ST-cDNA primer is shown in SEQ ID NO.

17.

3. The primer set for detecting Mycobacterium tuberculosis and rifampicin resistance according to claim 1, characterized in that: The 3' end of the sequence of SEQ ID NO. 8 is modified with an NH2-C6 group, the 3' end of the sequence of SEQ ID NO. 9 is modified with an NH2-C6 group, and the 5' end of the sequence of SEQ ID NO. 16 is modified with an SH group.

4. A kit for detecting Mycobacterium tuberculosis and rifampicin resistance, characterized in that: The primer set comprises the primer set for detecting Mycobacterium tuberculosis and rifampicin resistance according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that Including 2 volumes of 1 μM IS6110_F primer, IS6110_R primer, IS1081_F primer, IS1081_R primer, rpoB-F1 primer, rpoB-F2 primer, rpoB-F3 primer, rpoB-R primer, LNA-1 primer, LNA-2 primer, 5 volumes of PCR Buffer, 3 volumes of 25 mM MgCl2, 1 volume of 10 mM dNTP, 1 volume of 5 U / μL Taq DNA polymerase and 17 volumes of ddH2O.

6. A Mycobacterium tuberculosis and rifampicin resistance detection system, characterized in that: It includes genome sequence extraction module, PCR amplification module, mixed reaction module, Mycobacterium tuberculosis determination module and rifampicin resistance determination module; The genome sequence extraction module is used to extract the genome sequence of the sample to be tested; The PCR amplification module is used to perform multiple PCR amplification on the sample to be tested using the primer set according to any one of claims 1 to 3 or the kit according to claim 4 or 5; The mixed reaction module is used to denature the amplified product and perform a mixed reaction with the DNA "gate" structure composed of A1, A2 and L1, L2, L3 primers; The Mycobacterium tuberculosis determination module is used to mix the mixed reaction product with hemin, react with TMB color developing solution, and determine Mycobacterium tuberculosis based on color changes; The rifampicin resistance determination module is used to mix the mixed reaction product with the dsDNA-AuNPs solution incubated with ST-DNA and ST-cDNA and react, add NaCl solution after the reaction, and judge the rifampicin resistance of Mycobacterium tuberculosis based on the color change.

7. The Mycobacterium tuberculosis and rifampicin resistance detection system according to claim 6, characterized in that: The reaction procedure of the multiplex PCR amplification includes 95° C. for 5 min; 95° C. for 30 s, 55° C. for 35 s, and 72° C. for 1 min, 25 cycles; and 72° C. for 10 min.

8. The Mycobacterium tuberculosis and rifampicin resistance detection system according to claim 6, characterized in that: The reaction temperature of the mixed reaction is 37°C, and the reaction time is 30 min; the hemin concentration is 3 μM, the reaction temperature with the color developing solution is room temperature, the time is 10 min, and the amount of color developing solution added is 100 μL; the reaction temperature of the dsDNA-AuNPs solution incubated with ST-DNA and ST-cDNA and then mixed and reacted is 37°C, the reaction time is 30 min, the concentration of the NaCl solution is 0.2 M, and the volume is 100 μL.

9. A method for detecting Mycobacterium tuberculosis and rifampicin resistance, characterized in that: include: Extracting the genome sequence of the sample to be tested; Performing multiplex PCR amplification on the sample to be tested using the primer set according to any one of claims 1 to 3 or the kit according to claim 4 or 5; The amplified product is denatured and mixed with a DNA "gate" structure consisting of primers A1, A2 and L1, L2, and L3; The mixed reaction product is mixed with hemin, reacted with TMB color developing solution, and Mycobacterium tuberculosis is identified based on the color change; The mixed reaction product was mixed with the dsDNA-AuNPs solution incubated with ST-DNA and ST-cDNA and reacted. After the reaction, NaCl solution was added, and the rifampicin resistance of Mycobacterium tuberculosis was judged based on the color change.

10. Use of the primer set according to any one of claims 1 to 3 or the kit according to claim 4 or 5 in the preparation of a reagent for detecting rifampicin-resistant Mycobacterium tuberculosis.

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