Fluorescent quantitative PCR (Polymerase Chain Reaction) kit and method for detecting mycobacterium tuberculosis complex in tongue swab

Abstract

The invention relates to a fluorescent quantitative PCR kit and method for detecting a mycobacterium tuberculosis complex group in a tongue swab, and belongs to the field of pathogenic microorganism detection, the fluorescent quantitative PCR kit comprises the following components: an amplification reaction solution, a primer probe mixed solution and an external reference; the amplification reaction solution is a PCR (Polymerase Chain Reaction) buffer solution containing Taq DNA (Deoxyribose Nucleic Acid) polymerase, Mg < 2 + > ions, dNTP (dUTP) and UDG enzyme; the primer probe mixed solution comprises an IS6110 specific inner primer pair, an outer primer pair and a probe; the kit comprises an IS1081 specific inner primer pair, an IS1081 specific outer primer pair and a probe, a bacillus subtilis specific primer pair and a probe; the invention discloses a human gene specific primer pair and a probe. The fluorescent quantitative PCR kit disclosed by the invention can be used for rapidly and sensitively detecting the mycobacterium tuberculosis complex DNA in a tongue swab sample of a tuberculosis patient, has the advantages of high sensitivity and high specificity, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pathogen detection technology, specifically relating to a real-time PCR kit and method for detecting Mycobacterium tuberculosis complexes in tongue swabs. Background Technology

[0002] Tuberculosis is a respiratory infectious disease caused by infection with the Mycobacterium tuberculosis complex (MTBC), which mainly includes Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium africanum. Tuberculosis remains the leading cause of death from a single infectious disease worldwide. Early and accurate diagnosis and standardized treatment are beneficial for eliminating the source of infection and blocking the transmission chain.

[0003] However, traditional tuberculosis diagnostic techniques lack sufficient accuracy, resulting in 30%-40% of tuberculosis patients being missed: interpretation of imaging examinations (such as chest X-rays) is highly subjective and difficult to differentiate tuberculosis from other infectious or non-infectious lung diseases; the tuberculin skin test (TST) cannot distinguish between active tuberculosis, BCG vaccination, and non-tuberculous mycobacteria (NTM) infection due to antigen cross-reactivity, nor can it differentiate between latent tuberculosis infection and active tuberculosis; although sputum smear acid-fast staining is simple to perform, its sensitivity is low; culture methods, as the "gold standard" for tuberculosis diagnosis, are difficult to meet the clinical needs of early diagnosis and early treatment due to the long culture period of 6-8 weeks.

[0004] Traditional tuberculosis diagnosis usually uses sputum samples; however, approximately 25% of symptomatic and 90% of asymptomatic tuberculosis patients have difficulty expectorating. In addition, infectious aerosols are released during sputum collection, increasing the exposure risk to healthcare workers and other people being sampled, which is particularly prominent in primary healthcare institutions and large-scale active screening.

[0005] Tongue swabs are a new type of tuberculosis test sample. Using tongue swabs instead of sputum to diagnose tuberculosis has the following advantages: (1) Sampling is simple and can be collected from almost all tuberculosis patients, thus solving the diagnostic needs of patients with "little sputum / no sputum"; (2) It is not easy to generate aerosols, thus the biological risk is lower, and it can be carried out in primary medical institutions and active screening with limited protective measures.

[0006] Quantitative real-time PCR (qPCR) is a novel diagnostic technique that has been developed in the last ten years or so. This diagnostic technique has the following advantages: (1) Sensitivity: Due to the amplification of nucleic acid index, the detection signal is greatly improved, thus improving the detection of low-load bacteria; (2) Specificity: By detecting the specific gene targets of Mycobacterium tuberculosis complex, Mycobacterium tuberculosis complex and NTM can be directly distinguished; (3) Speed: Only a few tens of minutes of detection time is required, so anti-tuberculosis treatment can be started in time.

[0007] Directly detecting Mycobacterium tuberculosis complex nucleic acid in tongue swab samples is quite challenging, mainly because the bacterial load of Mycobacterium tuberculosis complex in tongue swab samples is low and there are interfering components. Therefore, no real-time quantitative PCR kits for detecting Mycobacterium tuberculosis complex in tongue swab samples have been reported to date. Summary of the Invention

[0008] To fill the gap in the detection of Mycobacterium tuberculosis complex in tongue swabs using real-time PCR, this invention provides a real-time PCR kit and method for detecting Mycobacterium tuberculosis complex in tongue swabs.

[0009] The technical solution adopted by this invention to solve the technical problem is as follows:

[0010] In a first aspect, the present invention provides a primer and probe system for detecting Mycobacterium tuberculosis complexes in tongue swabs, mainly comprising: an inner primer pair, an outer primer pair, and a probe for detecting the IS6110 sequence, and an inner primer pair, an outer primer pair, and a probe for detecting the IS1081 sequence.

[0011] Preferably, the nucleic acid sequences of the inner primer pair used to detect the IS6110 sequence are SEQ ID NO.1 and SEQ ID NO.2.

[0012] Preferably, the nucleic acid sequences of the outer primer pair used to detect the IS6110 sequence are SEQ ID NO.3 and SEQ ID NO.4.

[0013] Preferably, the nucleic acid sequence of the probe used to detect the IS6110 sequence is SEQ ID NO.5.

[0014] Preferably, the nucleic acid sequences of the inner primer pair used to detect the IS1081 sequence are SEQ ID NO.6 and SEQ ID NO.7.

[0015] Preferably, the nucleic acid sequences of the outer primer pair used to detect the IS1081 sequence are SEQ ID NO.8 and SEQ ID NO.9.

[0016] Preferably, the nucleic acid sequence of the probe used to detect the IS1081 sequence is SEQ ID NO.10.

[0017] The present invention provides a primer and probe system for detecting Mycobacterium tuberculosis complex in tongue swabs, and further includes primer pairs and probes for detecting Bacillus subtilis.

[0018] Preferably, the nucleic acid sequences of the primer pair used for detecting Bacillus subtilis are SEQ ID NO.11 and SEQ ID NO.12.

[0019] Preferably, the nucleic acid sequence of the probe used to detect Bacillus subtilis is SEQ ID NO.13.

[0020] The present invention provides a primer and probe system for detecting Mycobacterium tuberculosis complex in tongue swabs, and further includes primer pairs and probes for detecting human genes.

[0021] Preferably, the human-derived gene is GAPDH.

[0022] Preferably, the nucleic acid sequences of the primer pair used for detecting human genes are SEQ ID NO.14 and SEQ ID NO.15.

[0023] Preferably, the nucleic acid sequence of the probe used to detect human genes is SEQ ID NO.16.

[0024] Secondly, the present invention provides a real-time quantitative PCR kit for detecting Mycobacterium tuberculosis complexes in tongue swabs. The real-time quantitative PCR kit includes: an amplification reaction solution and a primer-probe mixture; the primer-probe mixture consists of a primer and probe system for detecting Mycobacterium tuberculosis complexes in tongue swabs.

[0025] Specifically, the amplification reaction solution contains Taq DNA polymerase and Mg. 2+ PCR buffer containing ions, dNTPs, and UDG enzymes.

[0026] Specifically, the primer-probe mixture includes an inner primer pair, an outer primer pair, and a probe for detecting the IS6110 sequence; an inner primer pair, an outer primer pair, and a probe for detecting IS1081; a primer pair and a probe for detecting human genes; and a primer pair and a probe for detecting Bacillus subtilis.

[0027] Specifically, the human-derived gene is GAPDH.

[0028] Thirdly, the present invention provides a quantitative real-time PCR method for detecting Mycobacterium tuberculosis complexes in tongue swabs. This quantitative real-time PCR method utilizes a quantitative real-time PCR kit for detecting Mycobacterium tuberculosis complexes in tongue swabs, as provided in the second aspect.

[0029] Specifically, the concentrations of each component in the PCR reaction system used in this quantitative real-time PCR detection method are as follows:

[0030] Taq DNA polymerase 0.065-0.085 U / μL; Mg 2+ Ions 4-6 mmol / L; dNTPs (dUTP) 0.25-0.45 mmol / L; UDG enzyme 0.00055-0.00075 U / μL; Inner primer pair for detecting IS6110 sequence 1-3 pmol / µL; Outer primer pair for detecting IS6110 sequence 1-3 pmol / µL; Probe for detecting IS6110 sequence 0.5-1.5 pmol / µL; Inner primer pair for detecting IS1081 sequence 1-3 pmol / µL; Outer primer pair for detecting IS1081 sequence 1-3 pmol / µL; Probe for detecting IS1081 sequence 0.5-1.5 pmol / µL; Primer pair for detecting human gene 0.5-1.5 pmol / µL; Probe for detecting human gene 0.05-0.15 pmol / µL; primer pairs for detecting Bacillus subtilis: 0.5-1.5 pmol / µL; probes for detecting Bacillus subtilis: 0.5-1.5 pmol / µL.

[0031] Specifically, the PCR amplification procedure used in this quantitative real-time PCR detection method is as follows:

[0032] (1) Digest UDG at 50℃ for 2 min;

[0033] (2) Preheat at 95℃ for 2 min;

[0034] (3) Pre-deformation at 95℃ for 10 s, annealing and extension at 65℃ for 30 s, repeated 5 times;

[0035] (4) Denaturation at 95℃ for 10 s, annealing and extension at 65℃ for 30 s (fluorescence collection), 40 cycles.

[0036] The beneficial effects of this invention are:

[0037] This invention, based on TaqMan probe-based real-time fluorescence PCR technology, develops a quantitative real-time PCR kit and method for detecting Mycobacterium tuberculosis complexes in tongue swabs. By using Bacillus subtilis as an external control, false negative results caused by PCR inhibitors in the sample or improper operation are monitored and avoided. The use of UNG enzyme effectively avoids contamination of amplification products. Simultaneous amplification of the multi-target sequences IS6110 and IS1081 increases the sensitivity of detection. The use of screened specific primers and nested PCR amplification ensures the specificity of the quantitative real-time PCR kit. Furthermore, the design of primers and probes with added human internal control genes allows for quality control of the sampling process, ensuring the accuracy of experimental results.

[0038] The fluorescence quantitative PCR kit of the present invention has the characteristics of high sensitivity and can detect H 37 The lower limit for Rv inactivation is 20 CFU / mL. The real-time PCR kit of this invention is highly specific, targeting only H... 37 The Rv-inactivated strains showed amplification signals, while Mycobacterium smegmatis, Mycobacterium avium, Mycobacterium terrestris, Mycobacterium kansas, Mycobacterium Asianum, Mycobacterium scrofula, Mycobacterium Gordonii, Mycobacterium guilloché, Mycobacterium occulta, Mycobacterium abscessus, Mycobacterium bufossa, Mycobacterium spp., and the negative control showed no amplification signals. The fluorescent quantitative PCR kit of the present invention has high clinical testing performance, with a sensitivity of 80% and a specificity of 100%. Attached Figure Description

[0039] Figure 1 This is the result of sensitivity testing. H 37 The concentrations of Rv inactivated bacterial solutions were 10000, 1000, 100, 50, and 20 CFU / mL, respectively, with physiological saline serving as a negative control.

[0040] Figure 2 This is a specific detection result. NTM-inactivated strains include *Mycobacterium smegmatis*, *Mycobacterium avium*, *Mycobacterium terrestris*, *Mycobacterium kansasense*, *Mycobacterium Asianum*, *Mycobacterium scrofula*, *Mycobacterium gordonii*, *Mycobacterium guildrums*, *Mycobacterium spp.*, *Mycobacterium abscessum*, *Mycobacterium bufo*, and *Mycobacterium spp.*, with a final concentration of 1×10⁻⁶. 6 CFU / mL, physiological saline as a negative control, H 37 Rv inactivated strains served as a positive control. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1: Composition and Preparation of the Reaction System for a Real-Time PCR Kit for Detecting Mycobacterium tuberculosis Complex in Tongue Swabs

[0043] 1. Design and screening of primers and probes;

[0044] Conserved regions of the Mycobacterium tuberculosis-specific IS6110 and IS1081 sequences were selected as targets for detection. Multiple sets of primers were designed for different sites in the IS1081 and IS6110 sequences, and each set of primers was tested and screened.

[0045] 2. Composition of the real-time PCR kit;

[0046] The real-time PCR kit prepared in Example 1 is mainly used to detect Mycobacterium tuberculosis complex in tongue swabs. It specifically includes the following components: amplification reaction solution and primer-probe mixture.

[0047] In Example 1, the amplification reaction solution specifically contains Taq DNA polymerase and Mg. 2+ PCR buffer containing ions, dNTPs (dUTPs), and UDG enzyme.

[0048] In Example 1, the primer-probe mixture consists of a primer and probe system for detecting the Mycobacterium tuberculosis complex in tongue swabs. Specifically, the primer and probe system for detecting the Mycobacterium tuberculosis complex in tongue swabs includes: IS6110 specific inner primer pair, IS6110 specific outer primer pair, and IS6110 specific probe; IS1081 specific inner primer pair, IS1081 specific outer primer pair, and IS1081 specific probe; Bacillus subtilis specific primer pair and Bacillus subtilis specific probe; and human gene specific primer pair and human gene specific probe.

[0049] Specifically, the nucleic acid sequences of the IS6110 specific inner primer pair are SEQ ID NO.1 and SEQ ID NO.2, the nucleic acid sequences of the IS6110 specific outer primer pair are SEQ ID NO.3 and SEQ ID NO.4, and the nucleic acid sequence of the IS6110 specific probe is SEQ ID NO.5; the nucleic acid sequences of the IS1081 specific inner primer pair are SEQ ID NO.6 and SEQ ID NO.7, the nucleic acid sequences of the IS1081 specific outer primer pair are SEQ ID NO.8 and SEQ ID NO.9, and the nucleic acid sequence of the IS1081 specific probe is SEQ ID NO.10; the nucleic acid sequences of the Bacillus subtilis specific primer pair are SEQ ID NO.11 and SEQ ID NO.12, and the nucleic acid sequence of the Bacillus subtilis specific probe is SEQ ID NO.13; the nucleic acid sequences of the human gene specific primer pair are SEQ ID NO.14 and SEQ ID NO.15, and the nucleic acid sequence of the human gene specific probe is SEQ ID NO.16. See Table 1.

[0050] Table 1

[0051]

[0052] 3. Preparation of the reaction mixture for real-time PCR;

[0053] Prepare the PCR reaction mixture by mixing 15 μL of amplification reaction solution with 3 μL of primer and probe mixture. Dispense 18 μL into each of eight-tube or PCR reaction tubes and perform fluorescent PCR amplification within 4 h.

[0054] The concentrations of each component in the PCR reaction mixture, i.e., the PCR reaction system, are as follows:

[0055] Taq DNA polymerase 0.075 U / μL; Mg 2+Ions 5 mmol / L; dNTPs (dUTP) 0.35 mmol / L; UDG enzyme 0.00065 U / μL; inner primer pair for detecting IS6110 sequence 2 pmol / µL; outer primer pair for detecting IS6110 sequence 2 pmol / µL; probe for detecting IS6110 sequence 1 pmol / µL; inner primer pair for detecting IS1081 sequence 2 pmol / µL; outer primer pair for detecting IS1081 sequence 2 pmol / µL; probe for detecting IS1081 sequence 1 pmol / µL; primer pair for detecting human gene 1 pmol / µL; probe for detecting human gene 0.1 pmol / µL; primer pair for detecting Bacillus subtilis 1 pmol / µL; probe for detecting Bacillus subtilis 1 pmol / µL.

[0056] Example 2: Method of using a real-time PCR kit for detecting Mycobacterium tuberculosis complexes in tongue swabs

[0057] 1. Tongue swab sample collection;

[0058] Do not eat, drink, or rinse your mouth for 1-2 hours before sampling; use a flocked swab to collect samples from the back of the tongue, on the front 2 / 3 of the tongue. Apply appropriate force during sampling, for 10-20 seconds, and swab at least 5 times before and after sampling; after collection, place the sample in 1 mL of sample preservation solution (sterile saline / TE buffer).

[0059] 2. Nucleic acid extraction;

[0060] Bacillus subtilis was added to the tongue swab sample as an external reference strain, and DNA was extracted using the magnetic bead method after thorough mixing.

[0061] 3. Add sample;

[0062] A negative control and a positive control were set up. The negative control was physiological saline, and the positive control was H2O. 37 Rv inactivated strain. 12 μL of the extracted nucleic acid solution was added to 18 μL of the PCR reaction mixture prepared in step 3 of Example 1, and the mixture was placed in a real-time quantitative PCR instrument for PCR amplification.

[0063] 4. Reaction procedure;

[0064] The PCR reaction system prepared in step 3 of Example 1 was used for real-time PCR detection. The above PCR reaction system is compatible with various real-time PCR instruments. This Example 2 mainly uses the SLAN-96S instrument as an example, with "module temperature control" selected for temperature control. The PCR amplification program settings are shown in Table 2. On the SLAN-96S instrument, the FAM channel, ROX channel, and CY5 channel were selected, and the fluorescence acquisition point was set to 65℃ for 30 s. In the "Parameter Settings" option of the SLAN-96S instrument, the amplification curve algorithm was selected as "relative fluorescence algorithm - log".

[0065] Table 2

[0066]

[0067] 5. Quality control and result reading;

[0068] After the PCR amplification program is completed, save the detection data file and set the parameters and analyze the results for the FAM, ROX and CY5 channels respectively. Specifically, (1) Baseline setting: The baseline can be set automatically (AutoBaseline) or adjusted according to the shape of the amplification curve. (2) Threshold setting: It should be higher than the highest point of the fluorescence value detected by the normal negative control. The fluorescence curve above the threshold should be an S-shaped amplification curve; otherwise, the experiment is invalid, and errors in the instrument, reagents and amplification conditions should be checked. After the detection is completed, the interpretation of the Mycobacterium tuberculosis complex results is shown in Table 3.

[0069] Table 3

[0070]

[0071] In Table 3, MTBC refers to the Mycobacterium tuberculosis complex.

[0072] Example 1: Sensitivity detection of a real-time PCR kit for detecting Mycobacterium tuberculosis complexes in tongue swabs.

[0073] Tongue swab samples were collected from healthy volunteers following step 1 of Example 2, and H was added. 37 Rv inactivation solution, making H 37 The final concentrations of the Rv inactivated bacterial solution were 10000, 1000, 100, 50, and 20 CFU / mL. Physiological saline was used as a negative control. Nucleic acid extraction was performed according to step 2 of Example 2. Sample addition, amplification reaction, quality control, and result reading were performed according to steps 3-5 of Example 2.

[0074] Sensitivity test results are as follows Figure 1As shown, the fluorescence quantitative PCR kit of the present invention detects H 37 The lower limit of Rv inactivation of strains is 20 CFU / mL, indicating that the real-time PCR kit of the present invention has high sensitivity.

[0075] Experimental Example 2: Specificity Detection of a Real-Time PCR Kit for Detecting Mycobacterium tuberculosis Complex in Tongue Swabs

[0076] Tongue swab samples were collected from healthy volunteers according to step 1 of Example 2, and NTM-inactivated strains (Mycobacterium smegmatis, Mycobacterium avium, Mycobacterium terrestris, Mycobacterium kansasii, Mycobacterium Asianum, Mycobacterium scrofula, Mycobacterium gordonii, Mycobacterium guildrums, Mycobacterium occulta, Mycobacterium abscessum, Mycobacterium bufo, Mycobacterium gracilis) were added to achieve a final concentration of 1×10⁻⁶. 6 CFU / mL, with physiological saline as a negative control, and H 37 The Rv inactivated strain was used as a positive control. Nucleic acid extraction was performed according to step 2 of Example 2, and sample addition, amplification reaction, quality control, and result reading were performed according to steps 3-5 of Example 2.

[0077] Specific test results such as Figure 2 As shown, the positive control is H. 37 Rv inactivated strains showed amplification signals, while NTM inactivated strains (Mycobacterium smegmatis, Mycobacterium avium, Mycobacterium terrestris, Mycobacterium kansas, Mycobacterium asiaticum, Mycobacterium scrofula, Mycobacterium gordonii, Mycobacterium guilloché, Mycobacterium occulta, Mycobacterium abscessus, Mycobacterium bufo, Mycobacterium spp.) and negative controls did not show amplification signals.

[0078] Experiment 3: Clinical application of a real-time PCR kit for detecting Mycobacterium tuberculosis complexes in tongue swabs.

[0079] The clinical performance of the present invention's real-time PCR kit was verified using tongue swab samples from 150 patients, including 55 patients with pulmonary tuberculosis and 95 patients without tuberculosis. All included tuberculosis patients were diagnosed by sputum culture or sputum molecular testing; all non-tuberculosis patients were diagnosed by clinicians with other diseases, and their sputum culture, sputum molecular testing, and other results were all negative.

[0080] The fluorescent quantitative PCR kit of the present invention was used to detect tongue swab samples from the above 150 patients. The detection results are shown in Table 4. 44 out of 55 patients with pulmonary tuberculosis were positive, with a sensitivity of 80%; 0 out of 95 patients without tuberculosis were positive, with a specificity of 100%.

[0081] Table 4

[0082]

[0083] In summary, the real-time PCR kit of the present invention can rapidly and sensitively detect Mycobacterium tuberculosis complex DNA in tongue swab samples from tuberculosis patients, and has the advantages of high sensitivity and high specificity, and has broad application prospects.

[0084] This invention discloses a real-time PCR kit and method for detecting Mycobacterium tuberculosis complexes in tongue swabs. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The product of this invention has been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the product described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

Claims

1. A primer and probe system for detecting Mycobacterium tuberculosis complexes in tongue swabs, characterized in that, include: Inner primer pairs, outer primer pairs, and probes for detecting the IS6110 sequence; inner primer pairs, outer primer pairs, and probes for detecting the IS1081 sequence. The nucleic acid sequences of the inner primer pair used to detect the IS6110 sequence are SEQ ID NO.1 and SEQ ID NO.2; The nucleic acid sequences of the outer primer pair used to detect the IS6110 sequence are SEQ ID NO.3 and SEQ ID NO.4; The nucleic acid sequence of the probe used to detect the IS6110 sequence is SEQ ID NO.5; The nucleic acid sequences of the inner primer pair used to detect the IS1081 sequence are SEQ ID NO.6 and SEQ ID NO.7; The nucleic acid sequences of the outer primer pair used to detect the IS1081 sequence are SEQ ID NO. 8 and SEQ ID NO. 9; The nucleic acid sequence of the probe used to detect the IS1081 sequence is SEQ ID NO.

10.

2. The primer and probe system for detecting Mycobacterium tuberculosis complex in tongue swabs according to claim 1, characterized in that, It also includes: primer pairs and probes for detecting Bacillus subtilis; the nucleic acid sequences of the primer pairs for detecting Bacillus subtilis are SEQ ID NO.11 and SEQ ID NO.12, and the nucleic acid sequence of the probe for detecting Bacillus subtilis is SEQ ID NO.

13.

3. The real-time PCR kit for detecting Mycobacterium tuberculosis complex in tongue swabs according to claim 1, characterized in that, It also includes: primer pairs and probes for detecting human genes; the human gene is GAPDH; the nucleic acid sequences of the primer pairs for detecting human genes are SEQ ID NO.14 and SEQ ID NO.15, and the nucleic acid sequence of the probes for detecting human genes is SEQ ID NO.

16.

4. A real-time PCR kit for detecting Mycobacterium tuberculosis complexes in tongue swabs, characterized in that, include: Amplification reaction solution and primer / probe mixture; The primer-probe mixture consists of a primer and probe system for detecting Mycobacterium tuberculosis complexes in tongue swabs.

5. The real-time PCR kit for detecting Mycobacterium tuberculosis complex in tongue swabs according to claim 4, characterized in that, The amplification reaction solution contains Taq DNA polymerase and Mg. 2+ PCR buffer containing ions, dNTPs, and UDG enzymes.

6. The real-time PCR kit for detecting Mycobacterium tuberculosis complex in tongue swabs according to claim 4, characterized in that, The primer-probe mixture includes inner primer pairs, outer primer pairs, and probes for detecting the IS6110 sequence, inner primer pairs, outer primer pairs, and probes for detecting IS1081, primer pairs and probes for detecting human genes, and primer pairs and probes for detecting Bacillus subtilis.

7. The real-time PCR kit for detecting Mycobacterium tuberculosis complex in tongue swabs according to claim 6, characterized in that, The human-derived gene is GAPDH.

8. A method for detecting Mycobacterium tuberculosis complex in tongue swabs using a real-time PCR kit for detecting Mycobacterium tuberculosis complex in tongue swabs as described in any one of claims 4-7.

9. The method for detecting Mycobacterium tuberculosis complex in tongue swabs according to claim 8, characterized in that, The concentrations of each component in the PCR reaction system used in this quantitative real-time PCR detection method are as follows: Taq DNA polymerase 0.065-0.085 U / μL; Mg 2+ Ions 4-6 mmol / L; dNTPs (dUTP) 0.25-0.45 mmol / L; UDG enzyme 0.00055-0.00075 U / μL; Inner primer pair for detecting IS6110 sequence 1-3 pmol / µL; Outer primer pair for detecting IS6110 sequence 1-3 pmol / µL; Probe for detecting IS6110 sequence 0.5-1.5 pmol / µL; Inner primer pair for detecting IS1081 sequence 1-3 pmol / µL; Outer primer pair for detecting IS1081 sequence 1-3 pmol / µL; Probe for detecting IS1081 sequence 0.5-1.5 pmol / µL; Primer pair for detecting human gene 0.5-1.5 pmol / µL; Probe for detecting human gene 0.05-0.15 pmol / µL; primer pairs for detecting Bacillus subtilis: 0.5-1.5 pmol / µL; probes for detecting Bacillus subtilis: 0.5-1.5 pmol / µL.

10. The method for detecting Mycobacterium tuberculosis complex in tongue swabs according to claim 8, characterized in that, The PCR amplification procedure used in this quantitative real-time PCR detection method is as follows: (1) Digest UDG at 50℃ for 2 min; (2) Preheat at 95℃ for 2 min; (3) Pre-deformation at 95℃ for 10 s, annealing and extension at 65℃ for 30 s, repeated 5 times; (4) Denaturation at 95℃ for 10 s, annealing and extension at 65℃ for 30 s (collect fluorescence), cycle 40 times.

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