Mycobacterium tuberculosis double-target joint detection kit and detection method

Abstract

The invention discloses a Mycobacterium tuberculosis double-target joint detection kit and a detection method, IS6110 and mpt64 genes are screened and determined as target genes for double-target joint detection of Mycobacterium tuberculosis, the detection false negative rate is reduced to a great extent, and the detection precision is improved. According to the invention, two pairs of specific primers and two probes are designed and synthesized, efficient, rapid and accurate double-target joint detection of mycobacterium tuberculosis is realized, the sensitivity and the specificity both reach 95% or more, and the matching degree of the two probes for joint detection of mycobacterium tuberculosis exceeds 90%. A unique reaction system is designed to carry out real-time fluorescent quantitative PCR, primer probes do not interfere with one another, amplification detection can be carried out at the same time, and high sensitivity and specificity are achieved. The sample does not need to be cultured, and the mycobacterium tuberculosis can be rapidly and accurately detected through sputum, so that the quantitative and qualitative detection of the mycobacterium tuberculosis is realized.

Description

(I) Technical field

[0001] The present invention relates to the technical field of biological detection, and in particular to a Mycobacterium tuberculosis dual-target combined detection kit and a detection method. (II) Background technology

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis complex (MTBC) infecting the lungs, posing a serious threat to human health.

[0003] The Mycobacterium tuberculosis complex includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum and other mycobacteria, all of which can cause pulmonary tuberculosis in humans.

[0004] At present, traditional clinical detection methods for Mycobacterium tuberculosis, such as alveolar lavage biopsy and culture methods, have problems such as cumbersome operation, long time consumption, and low sensitivity, and can no longer meet the needs of modern tuberculosis diagnosis. In comparison, molecular biological detection methods are less time-consuming, have lower detection limits, and higher specificity, becoming a new research direction to improve the level of tuberculosis diagnosis. However, the molecular biological detection of Mycobacterium tuberculosis on the market is mostly single-target gene detection, which has the problems of low specificity, high false negative rate, and the need to use alveolar lavage fluid.

[0005] Therefore, the development of a rapid, accurate and efficient method for detecting Mycobacterium tuberculosis is of great clinical significance. Currently, there is no reagent on the market that can perform dual-target gene molecular detection of Mycobacterium tuberculosis through sputum without the need for sputum culture. (III) Summary of the invention

[0006] The purpose of the present invention is to provide a dual-target combined detection kit and a detection method for Mycobacterium tuberculosis. The kit of the present invention can directly use sputum samples to detect Mycobacterium tuberculosis with high precision, which is rapid, accurate and efficient, and solves the problems of cumbersome operation, long time consumption, low sensitivity and high false negative rate of single gene target detection in traditional Mycobacterium tuberculosis detection methods.

[0007] The technical solution adopted by the present invention is:

[0008] The present invention provides a Mycobacterium tuberculosis dual-gene combined detection kit, the kit comprising primers and probes for detecting target gene IS6110 and target gene mpt64, a positive control, and a negative control; the primers include a forward primer and a reverse primer;

[0009] The sequences of the primers and probes are:

[0010] IS6110-F: 5'-GAAGAATCCGCTGAGATAAAGC-3' (SEQ ID NO. 1);

[0011] IS6110-R:5'-GGTTGATGTGGTCGTAGTAGGT-3' (SEQ ID NO.2);

[0012] IS6110-Probe:5'-TCACATAAGGCGTTGTCGAGGG-3'(SEQ ID NO.3);

[0013] mpt64-F:5'-GACTTCTGGTCGGGGTAGTAAC-3' (SEQ ID NO.4);

[0014] mpt64-R:5'-CTGTCGTTTTGCTCTGTTGTTC-3' (SEQ ID NO.5);

[0015] mpt64-Probe: 5'-TGTGGTGGCCGGCGTGTCCGCC-3' (SEQ ID NO. 6);

[0016] The 5' end of the probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1;

[0017] The positive controls are positive plasmids pMD18-T-IS6110 and pMV261-eGFP;

[0018] The negative control was a phosphate buffer solution of 0.067 mol / L and pH 6.8.

[0019] Further, the concentration of the positive plasmids was 10 4 copies / μL.

[0020] The design of the probe of the present invention follows the design rules of general fluorescent probe method.

[0021] The target gene IS6110 of the present invention is a multi-copy gene with high specificity, but it does not exist in all Mycobacterium tuberculosis; the target gene mpt64 has overlap with Mycobacterium cannae and cannot be relied upon alone for the detection of Mycobacterium tuberculosis. The present invention combines the two target genes for the detection of Mycobacterium tuberculosis, thereby improving the accuracy and sensitivity of Mycobacterium tuberculosis detection.

[0022] Furthermore, the kit also includes a PCR reaction solution, which includes dNTPs, Tris-HCl at pH 8 to 9, MgCl2, DNA polymerase, KCl, ammonium sulfate, tetramethylammonium chloride (TMAC), a nuclease inhibitor, and UDG enzyme.

[0023] Furthermore, the composition of 50 μL of PCR reaction solution is: 50 μM each of IS6110 forward and reverse primers, mpt64 forward and reverse primers, 25 μM each of IS6110 probe and mpt64 probe, 50 μM each of fluorescent group 6-FAM and quenching group BHQ1, 300 μM of dNTPs, 5 mM of MgCl2, 35 mM of KCl, 10 mM of ammonium sulfate, 60 mM of TMAC, 25 U of DNA polymerase, 1.3 U of nuclease inhibitor (DEPC), 0.03 U of UDG enzyme, 5 μL of cDNA template, and finally, the reagents are supplemented to 50 μL with pH = 8-9, 30 mM Tris-HCl buffer.

[0024] The present invention provides a method for detecting Mycobacterium tuberculosis using a Mycobacterium tuberculosis double gene combined detection kit, wherein the method comprises the following steps:

[0025] (1) Preparation of the sample to be tested: After pre-treating the sputum to be tested by the N-acetyl-L-cysteine-sodium hydroxide method, genomic DNA is extracted (preferably, the whole genomic DNA of Mycobacterium tuberculosis in the sample to be tested is extracted according to the instruction manual of the GF-1 tissue DNA extraction kit), and cDNA of the sample to be tested is prepared;

[0026] (2) 50 μL PCR reaction solution contains: IS6110 forward and reverse primers, mpt64 forward and reverse primers, 50 μM each, IS6110 probe, mpt64 probe, 25 μM each, fluorescent group 6-FAM, quenching group BHQ1, 50 μM each, dNTPs 300 μM, MgCl2 5 mM, KCl 35 mM, ammonium sulfate 10 mM, TMAC 60 mM, DNA polymerase 25 U, nuclease inhibitor (DEPC) 1.3 U, UDG enzyme 0.03 U, cDNA template 5 μL, and finally 30 mM Tris-HCl (pH = 8-9) is used to make up the reagents to 50 μL;

[0027] PCR amplification: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 30 sec, denaturation at 60°C for 40 sec, annealing at 72°C for 40 sec, 40 cycles;

[0028] (3) Result interpretation: If ct ≤ 30, the result is positive; if ct value > 30 or no ct value is found, the result is negative.

[0029] Furthermore, the sputum pretreatment method for the test in step (1) is as follows: the sputum sample is mixed with an equal volume of 1% NALC-NaOH, incubated at 20-25° C. for 15 minutes, 9 volumes of phosphate buffer (0.067 mol / L, pH 6.8) are added to the mixture, centrifuged at 3000×g for 15 minutes, the supernatant is discarded, the precipitate is redissolved with distilled water, and the mixture is bathed in a water bath at 80° C. for 40 minutes to kill bacteria, and centrifuged at 8000×g for 1 minute. The precipitate is the sample after pretreatment.

[0030] Further, the 1% NALC-NaOH is prepared by mixing a 4% NaOH aqueous solution and a 2.94% sodium citrate aqueous solution in a volume ratio of 7:3 to obtain a basic solution, adding N-acetyl-L-cysteine ​​to the basic solution to make the concentration of N-acetyl-L-cysteine ​​10 g / L, and adjusting the pH between 2.0 and 2.8 to obtain 1% NALC-NaOH.

[0031] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0032] 1. According to the different characteristics of each gene in Mycobacterium tuberculosis, the present invention screened and determined the IS6110 and mpt64 genes as the target genes for dual-target combined detection of Mycobacterium tuberculosis, which greatly reduced the false negative rate of detection and improved the detection accuracy.

[0033] 2. The present invention designs and synthesizes two pairs of specific primers and two probes for the detection determining regions of the IS6110 and mpt64 genes, realizing efficient, rapid and accurate dual-target joint detection of Mycobacterium tuberculosis, with a sensitivity and specificity of more than 95%. At the same time, the matching degree of the two probes for the joint detection of Mycobacterium tuberculosis exceeds 90%.

[0034] 3. The present invention designs a unique reaction system for real-time fluorescence quantitative PCR. There is no mutual interference between the primer probe pairs, and amplification detection can be performed simultaneously with high sensitivity and specificity. The sample does not need to be cultured, and Mycobacterium tuberculosis can be detected quickly and accurately through sputum, realizing quantitative and qualitative detection of Mycobacterium tuberculosis. (IV) Description of the drawings

[0035] Figure 1 The present invention is a flowchart of the use of the Mycobacterium tuberculosis double gene combined detection kit.

[0036] Figure 2 The positive and negative detection results of the IS6110 probe for Mycobacterium tuberculosis in the FAM channel of Example 2 (the relationship curve between the ct value and the Rn value).

[0037] Figure 3The positive and negative detection results of the mpt64 probe for Mycobacterium tuberculosis in the FAM channel of Example 2 (the relationship curve between the ct value and the Rn value). (V) Specific implementation methods

[0038] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0039] Example 1. Design of specific primers and probes for Mycobacterium tuberculosis

[0040] 1. Primer and probe design

[0041] According to the IS6110 gene sequence of Mycobacterium tuberculosis (Gene ID: 885699 on the official website of NCBI) and the mpt64 gene sequence (Gene ID: 885925 on the official website of NCBI), through the official website of Integrated DNA Technologies, select Primer Quest Tool in the TOOLS option in the service column, input the sequence containing the known target gene fragment, and select qPCR (2primers+probe; for use in 5'nuclease assays) for primer and probe design;

[0042] The design of primers and probes must meet the following conditions:

[0043] (I) Primers are designed on both the upstream and downstream sides of the detection probe to ensure the specificity, conservatism and inclusiveness of the primers.

[0044] (II) The probe was designed downstream of the forward primer and upstream of the reverse primer, with a 100% match to the wild-type gene.

[0045] (III) The probe can cover all sites of the corresponding detection gene segment. The 5' end of the probe is connected to the fluorescent group 6-FAM, and the 3' end is connected to the quenching group BHQ1. The probe can also be modified in various types according to needs.

[0046] (IV) The probe must meet the following requirements: the (G+C) content of the bases is between 40% and 60%, the base sequence does not form dimers, there is no complementary region, and the Tm is ≥ 55°C when hybridizing with the template chain.

[0047] (V) Probes and primers follow the general design rules for primers and probes.

[0048] 2. Initial screening

[0049] Step 1 Preliminary screening resulted in more than 100 pairs of probe-primer combinations. Using a DNA synthesizer (Beijing Haijing High-Tech Co., Ltd.), more than 100 probes and primers were synthesized by the solid phase phosphite triester method.

[0050] 3. Rescreening

[0051] According to Table 1, a real-time fluorescence PCR system was constructed and the positive samples (concentration 10 4 The positive plasmids pMD18-T-IS6110 and pMV261-eGFP (200 copies / μL) and negative samples (phosphate buffer 0.067 mol / L, pH 6.8) were added into the positive sample tube and negative sample tube set up in the eight-tube strip, respectively.

[0052] More than 100 pairs of probes and primers synthesized above were added into eight tubes respectively, and a PCR reaction system was configured to perform a qPCR real-time fluorescence reaction.

[0053] Table 1. Real-time fluorescence quantitative PCR reaction system for probe and primer screening

[0054]

[0055] Place the eight-tube strip into the PCR instrument, edit the reaction program and run it. The PCR reaction program is:

[0056] Step 1: denaturation at 94°C for 10 min;

[0057] Step 2: denaturation at 94°C for 30 seconds;

[0058] Step 3: denaturation at 60°C for 40s;

[0059] Step 4: Denaturation at 72°C for 40 seconds;

[0060] Step 5: The second, third and fourth steps of the above reaction were repeated 40 times;

[0061] Step 6: Collect the light signal at 42°C and excitation wavelength of 493nm;

[0062] Step 7: Arrange the light signal values ​​obtained from the above experiments, compare the data, and perform screening. The final sequences of the primers and probes obtained by screening are

[0063] IS6110-F: 5'-GAAGAATCCGCTGAGATAAAGC-3' (SEQ ID NO. 1); IS6110-R: 5'-GGTTGATGTGGTCGTAGTAGGT-3' (SEQ ID NO. 2); IS6110-Probe: 5'-TCACATAAGGCGTTGTCGAGGG-3' (SEQ ID NO. 3);

[0064] mpt64-F:5'-GACTTCTGGTCGGGGTAGTAAC-3' (SEQ ID NO.4);

[0065] mpt64-R:5'-CTGTCGTTTTGCTCTGTTGTTC-3' (SEQ ID NO.5);

[0066] mpt64-Probe: 5'-TGTGGTGGCCGGCGTGTCCGCC-3' (SEQ ID NO. 6).

[0067] The primers and probes were synthesized by the solid phase phosphite triester method using a DNA synthesizer (Beijing Haijing Gaochuang Technology Co., Ltd.).

[0068] Example 2: Plotting of FAM channel curves for detecting negative and positive samples using specific probes

[0069] 1. Drawing of FAM channel curves for negative and positive samples detected by IS6110 probe

[0070] (1) Construction of real-time fluorescence PCR system

[0071] Set up the eight-tube strips as positive sample tubes and negative sample tubes, prepare the PCR reaction system according to Table 2, and add the positive sample (concentration 10 4 The positive plasmids pMD18-T-IS6110 and pMV261-eGFP (200 copies / μL) and negative samples (phosphate buffer 0.067 mol / L, pH 6.8) were added into the positive sample tube and negative sample tube set up in the eight-tube strip, respectively.

[0072] Table 2. IS6110 specific probe real-time fluorescence quantitative PCR reaction system

[0073]

[0074] (2) Plotting FAM channel curves for negative and positive samples

[0075] Place the eight-tube strip into the PCR instrument, edit the reaction program and run it. The PCR reaction program is:

[0076] Step 1: denaturation at 94°C for 10 min;

[0077] Step 2: denaturation at 94°C for 30 seconds;

[0078] Step 3: denaturation at 60°C for 40s;

[0079] Step 4: Denaturation at 72°C for 40 seconds;

[0080] Step 5: The second, third and fourth steps of the above reaction were repeated 40 times;

[0081] Step 6: Collect the light signal at 42°C and excitation wavelength of 493nm;

[0082] Step 7: Arrange the optical signal values ​​obtained from the above experiments and draw the FAM channel curves of the IS6110 specific probe to detect negative and positive samples, respectively, as shown in Figure 2. Figure 2 shown.

[0083] 2. Plotting of FAM channel curves of negative and positive samples detected by mpt64 probe

[0084] (1) Construction of real-time fluorescence PCR system

[0085] Set up the eight-tube strips as positive sample tubes and negative sample tubes, prepare the PCR reaction system according to Table 3, and add the positive sample (concentration 10 4 The positive plasmids pMD18-T-IS6110 and pMV261-eGFP (200 copies / μL) and negative samples (phosphate buffer 0.067 mol / L, pH 6.8) were added into the positive sample tube and negative sample tube set up in the eight-tube strip, respectively.

[0086] Table 3. Real-time fluorescence quantitative PCR reaction system of mpt64 specific probe

[0087]

[0088]

[0089] (2) Plotting FAM channel curves for negative and positive samples

[0090] Place the eight-tube strip into the PCR instrument, edit the reaction program and run it. The PCR reaction program is:

[0091] Step 1: denaturation at 94°C for 10 min;

[0092] Step 2: denaturation at 94°C for 30 seconds;

[0093] Step 3: denaturation at 60°C for 40s;

[0094] Step 4: Denaturation at 72°C for 40 seconds;

[0095] Step 5: The second, third and fourth steps of the above reaction were repeated 40 times;

[0096] Step 6: Collect the light signal at 42°C and excitation wavelength of 493nm;

[0097] Step 7: Arrange the light signal values ​​obtained from the above experiments and draw the FAM channel curves of the mpt64-specific probe to detect negative and positive samples, respectively, as shown in Figure 2. Figure 3 shown.

[0098] from Figure 2 , Figure 3 It can be seen that the probe has significant specificity.

[0099] Example 3: Method for detecting Mycobacterium tuberculosis by real-time fluorescence PCR using a dual-target combined detection kit for Mycobacterium tuberculosis

[0100] 1. Sample source

[0101] Inactivated sputum samples (containing IS6110 and mpt64 genes) from patients with Mycobacterium tuberculosis were obtained in the laboratory of a hospital. The collection of samples followed medical ethical standards.

[0102] 2. Sample pretreatment

[0103] (1) Preparation of 1% NALC-NaOH:

[0104] ① Preparation of basic solution: Mix 350 mL of 4% NaOH aqueous solution and 150 mL of 2.94% sodium citrate aqueous solution in a volume ratio of 7:3 to obtain 500 mL of basic solution.

[0105] ② Add N-acetyl-L-cysteine: Add 5 g of N-acetyl-L-cysteine ​​to 500 mL of the above base solution.

[0106] ③ Adjust the pH value: Use pH test paper or pH meter to monitor the pH value of the solution in step ②, and use NaOH or dilute acid (such as dilute hydrochloric acid) to adjust the pH to between 2.0 and 2.8 to obtain 1% NALC-NaOH.

[0107] (2) Sample pretreatment

[0108] Place 2.5 mL of sputum sample in a 50 mL centrifuge tube and add 2.5 mL of 1% NALC-NaOH. Then close the centrifuge tube and shake it, incubate it at 20-25°C for 15 minutes. After that, add phosphate buffer (0.067 mol / L, pH 6.8) to 45 mL in the centrifuge tube. Centrifuge at 3000xg for 15 minutes. After the precipitation process is completed, carefully discard the supernatant into a sterile bottle, and re-dissolve the remaining precipitate with 1 mL of distilled water, which is the sample after purification and culture, and transfer it to a 1.5 mL microcentrifuge tube.

[0109] 3. Extraction of sample DNA

[0110] Place the microcentrifuge tube from step 2 in a water bath at 80°C for 40 minutes to kill bacteria. Centrifuge at 8000 x g for 1 min. Carefully remove the supernatant. Use the GF-1 Tissue DNA Extraction Kit (GF-TD-100, Vivantis, Selangor, Malaysia) to extract genomic DNA from the precipitate as a template. For specific procedures, refer to the kit instructions.

[0111] 4. Setting up negative and positive control groups

[0112] Positive control group: Amplify the nucleotide fragment containing the IS6110 target gene sequence by conventional PCR method, clone the amplified product into plasmid pMD18-T according to conventional gene cloning operation, and transform Escherichia coli to obtain recombinant plasmid pMD18-T-IS6110. 4 The positive plasmids (pMD18-T-IS6110 and pMV261-eGFP) with 10 copies / μL were used as positive controls.

[0113] Negative control group: Spotting buffer (phosphate buffer 0.067 mol / L, pH 6.8) was used as the negative control group.

[0114] 5. Construction of real-time fluorescence quantitative PCR reaction system:

[0115] Set up a positive control tube, a negative control tube, and a sample tube in the eight-tube strip, prepare the PCR reaction system according to Table 3, and add the positive control, negative control, and sample DNA into the positive control tube, negative control tube, and sample tube set up in the eight-tube strip, respectively.

[0116] Table 3 Real-time fluorescence quantitative PCR reaction system

[0117]

[0118]

[0119] 6. Specificity detection

[0120] Place the eight-tube strip into the PCR instrument, edit the reaction program and run it. The PCR reaction program is:

[0121] Step 1: denaturation at 94°C for 10 min;

[0122] Step 2: denaturation at 94°C for 30 seconds;

[0123] Step 3: denaturation at 60°C for 40s;

[0124] Step 4: Denaturation at 72°C for 40 seconds;

[0125] Step 5: The second, third and fourth steps of the above reaction were repeated 40 times;

[0126] Step 6: Collect light signals at 42°C and an excitation wavelength of 493nm.

[0127] 7. Test results:

[0128] The light signal values ​​obtained from the above experiments were sorted out, and the FAM channel amplification curves of the detection results of each tube were plotted. The FAM channel amplification curve corresponding to the sample tube was compared with the FAM channel amplification curves corresponding to the positive control tube and the negative control tube to determine whether MTBC exists in the test sample.

[0129] 8. Result judgment:

[0130] If the ct value in the sample tube is ≤30, the result is positive; if the ct value is >30 or there is no ct value, the result is negative.

[0131] Example 4: Accuracy of the Mycobacterium tuberculosis dual-target combined detection kit

[0132] Clinical samples: patients with pulmonary tuberculosis, lung cancer, nontuberculous mycobacterial infection, fungal infection and other easily confused diseases in a hospital in Zhejiang Province, the sample type is sputum. A total of 1183 clinical samples were enrolled, including 241 positive samples and 942 negative samples. The collection of samples complies with the patient's informed consent, protection of patient privacy and medical ethics requirements. The method of Example 3 was used for detection, and the results are shown in Table 4.

[0133] Bronchoalveolar lavage biopsy was used for comparison.

[0134] Table 4 Results of MTBC detection by two methods

[0135]

[0136] The experimental results show that for the detection of Mycobacterium tuberculosis, the kit of the present invention detected 237 positive cases, with a positive rate of 98.3%, and 946 negative cases, with a negative rate of 100.4%. The positive coincidence rate of the kit of the present invention and the clinical alveolar lavage biopsy test results is 97.10%, and the negative coincidence rate is 99.68%. In summary, the product has good consistency with the comparison method, and its performance can meet clinical needs.

[0137] As can be seen from the above embodiments, the present invention provides a preparation and application of primers, probes and detection reagents for the combined detection of Mycobacterium tuberculosis in sputum using dual-target genes. The reagents include primer probe sets of IS6110 and mpt64. The present invention designs and optimizes specific primers and fluorescent probes for Mycobacterium tuberculosis, and realizes efficient, rapid and accurate dual-target combined detection of Mycobacterium tuberculosis. The reagent of the present invention can perform qualitative detection of Mycobacterium tuberculosis in sputum without in vitro bacterial culture, thereby assisting clinicians in the diagnosis of pulmonary tuberculosis and improving medical efficiency. At the same time, the primer probe combination designed by the present invention has high sensitivity and specificity.

[0138] The above content is only an implementation scheme of the present invention. For technical researchers in this field, without departing from the purpose and scope of the present invention and without conducting unnecessary experiments, the results of the present invention can be implemented under the same parameters and temperature conditions, and several improvements and modifications can be made. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A Mycobacterium tuberculosis dual-gene combined detection kit, characterized in that: The kit includes primers, probes, positive controls, and negative controls for detecting target gene IS6110 and target gene mpt64; the primers include forward primers and reverse primers; The sequences of the primers and probes are: IS6110-F: 5'-GAAGAATCCGCTGAGATAAAGC-3'; IS6110-R:5'-GGTTGATGTGGTCGTAGTAGGT-3'; IS6110-Probe:5'-TCACATAAGGCGTTGTCGAGGG-3'; mpt64-F:5'-GACTTCTGGTCGGGGTAGTAAC-3'; mpt64-R:5'-CTGTCGTTTTGCTCTGTTGTTC-3'; mpt64-Probe:5'-TGTGGTGGCCGGCGTGTCCGCC-3'; The 5' end of the probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1; The positive controls are positive plasmids pMD18-T-IS6110 and pMV261-eGFP; The negative control was a phosphate buffer solution of 0.067 mol / L and pH 6.

8.

2. The kit according to claim 1, characterized in that The concentration of the positive plasmids was 10 4 copies / μL.

3. The kit according to claim 1, characterized in that The kit also includes a PCR reaction solution, which includes dNTPs, Tris-HCl at pH 8 to 9, MgCl2, DNA polymerase, KCl, ammonium sulfate, tetramethylammonium chloride, a nuclease inhibitor, and UDG enzyme.

4. The kit according to claim 3, characterized in that The composition of 50μL PCR reaction solution is: 50μM each of IS6110 forward and reverse primers, mpt64 forward and reverse primers, 25μM each of IS6110 probe and mpt64 probe, 50μM each of fluorescent group 6-FAM and quenching group BHQ1, 300μM dNTPs, 5mM MgCl2, 35mM KCl, 10mM ammonium sulfate, 60mM tetramethylammonium chloride, 25U DNA polymerase, 1.3U nuclease inhibitor, 0.03U UDG enzyme, 5μL cDNA template, and finally use pH=8-9, 30mM Tris-HCl buffer to make up the reagents to 50μL.

5. A method for detecting Mycobacterium tuberculosis using the Mycobacterium tuberculosis double gene combined detection kit according to claim 1, characterized in that: The method comprises the following steps: (1) Preparation of test samples: After pre-treatment of the test sputum using the N-acetyl-L-cysteine-sodium hydroxide method, genomic DNA was extracted to prepare cDNA of the test sample; (2) 50 μL PCR reaction solution contains: IS6110 forward and reverse primers, mpt64 forward and reverse primers 50 μM each, IS6110 probe, mpt64 probe 25 μM each, fluorescent group 6-FAM, quenching group BHQ1 50 μM each, dNTPs 300 μM, MgCl2 5 mM, KCl 35 mM, ammonium sulfate 10 mM, tetramethylammonium chloride 60 mM, DNA polymerase 25 U, nuclease inhibitor 1.3 U, UDG enzyme 0.03 U, cDNA template 5 μL, and finally use pH = 8-9, 30 mM Tris-HCl buffer to make up the reagents to 50 μL; (3) PCR amplification: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 30 sec, denaturation at 60°C for 40 sec, annealing at 72°C for 40 sec, 40 cycles; (4) Result interpretation: If ct ≤ 30, the result is positive; if ct value > 30 or no ct value is found, the result is negative.

6. The method according to claim 5, characterized in that The method for pre-treatment of the sputum to be tested in step (1) is as follows: the sputum sample is mixed with an equal volume of 1% NALC-NaOH, incubated at 20-25° C. for 15 minutes, 9 times the volume of 0.067 mol / L, pH 6.8 phosphate buffer is added to the mixture, centrifuged at a centrifugal force of 3000×g for 15 minutes, the supernatant is discarded, the precipitate is redissolved with distilled water, the mixture is bathed in a water bath at 80° C. for 40 minutes to kill bacteria, and the mixture is centrifuged at a centrifugal force of 8000×g for 1 minute. The precipitate is the sample after pre-treatment.

7. The method according to claim 6, characterized in that The 1% NALC-NaOH is prepared by mixing a 4% NaOH aqueous solution and a 2.94% sodium citrate aqueous solution in a volume ratio of 7:3 to obtain a basic solution, adding N-acetyl-L-cysteine ​​to the basic solution to make the concentration of N-acetyl-L-cysteine ​​10 g / L, and adjusting the pH between 2.0 and 2.8 to obtain 1% NALC-NaOH.

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