Mycobacterium tuberculosis detection and activity determination method based on fluorescent T7-CRISPR

Abstract

The invention belongs to the technical field of biology, and relates to a pathogen detection technology based on a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, in particular to a tubercle bacillus detection and activity determination method based on fluorescent T7-CRISPR. The first purpose is to provide a reagent combination for detecting viable tubercle bacillus, and the reagent combination comprises a) a T7 reverse transcription reaction reagent and b) a CRISPR-Cas13a reaction system; a) the T7 reverse transcription reaction reagent comprises a T7 reverse transcription primer and a T7 reverse transcriptase; and b) the CRISPR-Cas13a reaction system comprises specific crRNA, a fluorescence labeled ssRNA reporter, a reaction buffer solution, a Cas13a protein, RNase-Free H2O and a T7 reverse transcriptase. The invention also provides application of the reagent combination in detection of viable tubercle bacillus. The primer combination disclosed by the invention has the advantages of strong specificity and high sensitivity in detection application, can realize rapid detection and visual detection, and is low in detection cost, simple and convenient to operate and suitable for large-scale clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to pathogen detection technology based on the CRISPR system, specifically to a method for detecting and determining the activity of Mycobacterium tuberculosis based on fluorescent T7-CRISPR. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis, posing a serious threat to human health. Traditional TB detection methods, such as smear microscopy and culture, suffer from low sensitivity and long processing times. While nucleic acid amplification-based methods like PCR offer high sensitivity, they require expensive equipment and cannot distinguish between live and dead bacteria, easily leading to false positives. Currently, the EF-Tu mRNA molecule, unique to live bacteria, has an extremely short half-life and degrades rapidly after the death or dormancy of TB bacteria, making it an ideal target for detecting bacterial viability. However, traditional nucleic acid detection methods typically require specialized equipment such as quantitative real-time PCR instruments, which are complex and costly, unsuitable for primary healthcare units and rapid on-site testing.

[0003] Specifically, the main methods for detecting tuberculosis bacteria currently include the following: Smear microscopy: This is one of the most commonly used methods for detecting tuberculosis. It involves observing acid-fast bacilli in sputum using an optical or fluorescence microscope. While this method is simple, inexpensive, and rapid, its sensitivity is relatively low; a bacterial abundance of 5,000-10,000 per milliliter of sputum is typically required to detect a positive result. Furthermore, smear microscopy cannot distinguish between live and dead bacteria, nor can it differentiate between active tuberculosis bacilli, i.e., whether they are viable.

[0004] Culture method: The culture method is the "gold standard" for detecting tuberculosis bacteria, with high sensitivity, but it is time-consuming (usually requiring several weeks). The culture method can distinguish between live and dead bacteria, but the operation is complex and requires specialized laboratory equipment and personnel.

[0005] PCR technology: While nucleic acid amplification-based PCR technology has high sensitivity, it cannot distinguish between live and dead bacteria, easily leading to false positive results. Furthermore, PCR testing requires expensive equipment and skilled personnel, making it unsuitable for primary healthcare facilities and rapid on-site testing.

[0006] Immunological testing: This method determines the infection status by detecting tuberculosis-specific antigens or antibodies. For example, detecting the MPB64 antigen can be used for the immunological detection of tuberculosis, but this method requires sophisticated sample processing and cannot directly distinguish between live and dead bacteria.

[0007] Loop-mediated isothermal amplification (LAMP): LAMP is a rapid nucleic acid amplification method that obtains results in a short time by performing the amplification reaction at 67°C. However, LAMP also cannot distinguish between live and dead bacteria, and it requires certain equipment and is relatively expensive.

[0008] In summary, existing tuberculosis detection methods have the following shortcomings: Inability to distinguish between live and dead bacteria: Traditional detection methods (such as smear microscopy, culture, PCR, etc.) cannot accurately distinguish the activity state of Mycobacterium tuberculosis, which can easily lead to false positive results and affect the accuracy of clinical diagnosis.

[0009] Low detection sensitivity: Traditional methods have limited sensitivity for detecting Mycobacterium tuberculosis and are difficult to detect low concentrations of Mycobacterium tuberculosis, especially in early infection or low viral load samples.

[0010] The detection process is time-consuming: Traditional detection methods such as culture methods are time-consuming and cannot meet the needs of rapid diagnosis.

[0011] Reliance on complex equipment: Nucleic acid amplification-based methods (such as PCR) require expensive instruments (such as quantitative real-time PCR instruments), are complex to operate, and have high costs, making them unsuitable for primary healthcare units and rapid on-site testing.

[0012] Lack of visual detection methods: Most existing detection methods require specialized equipment and complex operations, making it impossible to achieve intuitive and rapid on-site detection.

[0013] In current clinical testing, detecting the viability of Mycobacterium tuberculosis remains a technical challenge. This is because, while international research indicates that EF-Tu mRNA is a biomarker specific to viable Mycobacterium tuberculosis, its extremely short half-life makes it difficult to detect using current commercially available technologies. In recent years, CRISPR-Cas13a technology, as a crRNA-guided RNA-targeting nuclease system, has attracted considerable attention due to its high specificity (single-base mismatch recognition) and signal amplification capabilities, requiring no complex sample pretreatment and allowing direct application in nucleic acid detection. Furthermore, T7 reverse transcriptase technology converts DNA sequences into complementary RNA through reverse transcription, providing a template for subsequent nucleic acid amplification and detection. The in vitro transcription process, initiated by T7 RNA polymerase, amplifies the nucleic acid signal and improves detection sensitivity. However, currently, there is a lack of a viable bacterial visualization detection method based on a single target of EF-Tu mRNA combined with T7-CRISPR technology. Therefore, developing a rapid, simple, and accurate method for detecting Mycobacterium tuberculosis and determining its viability is of significant clinical importance and application value. Summary of the Invention

[0014] To address the technical problems of existing technologies that cannot distinguish between live and dead Mycobacterium tuberculosis, and that are complex and time-consuming, this invention combines T7 reverse transcription technology with the CRISPR-Cas13a system to achieve rapid and intuitive detection using fluorescence signals.

[0015] The first object of the present invention is to provide a reagent combination for detecting live Mycobacterium tuberculosis, the reagent combination comprising: a) T7 reverse transcription reagent; b) CRISPR-Cas13a reaction system; a) The T7 reverse transcription reaction reagent includes a T7 promoter primer and a T7 reverse transcriptase; The T7 promoter primer is: T7-1F: 5'-TAATACGACTCACTATAGGTGACACTGCCGGAGGGCACCGAGATGGTGA-3' (SEQ IDNO.1) T7-1R: 5'-TAATACGACTCACTATAGTGATGATCTTGGTGACCCGGCCGGCGCCCAC-3' (SEQ IDNO.2) T7-2F: 5'-TAATACGACTCACTATAGGGCACCGAGATGGTGATGCCCGGTGACAACA-3' (SEQ IDNO.5) T7-2R: 5'-TAATACGACTCACTATAGCTTGATGATCTTGGTGACCCGGCCGGCGCCC-3' (SEQ IDNO.6) T7-3F: 5'-TAATACGACTCACTATAGCACCGACGTGACCGGTGTGTGTGACACTGCCG-3' (SEQ IDNO.7) T7-3R: 5'-TAATACGACTCACTATAGCTACTTGATGATCTTGGTGACCCCGGCCGGCG-3' (SEQ IDNO.8) b) The CRISPR-Cas13a reaction system includes specific crRNA and fluorescently labeled ssRNA reporter: The specific crRNA has the nucleotide sequence shown in SEQ ID NO.3: 5'-uaauuucuacuaaguguagauGCGAUCCGCGAGGGUGGCCGCAC-3; The fluorescently labeled ssRNA reporter has the following nucleotide sequence: 5'-CAL Fluor® Red 610-UUUUUU-(Int BHQ2 dT)-3'.

[0016] The CAL Fluor® Red 610 is a fluorescent dye, and the Int BHQ2 dT chain contains a deoxythymidine with a BHQ2 quenching group inserted into it.

[0017] Furthermore, b) the CRISPR-Cas13a reaction system also includes a reaction buffer, Cas13a protein, RNase-Free H2O, and T7 reverse transcriptase.

[0018] Furthermore, the CRISPR-Cas13a reaction system contains: 2 μL 10× reaction buffer, 0.5 μL 200 μM Cas13a protein, 0.5 μL 200 μM crRNA, 3 μL 100 μM ssRNA reporter, 1 μL 50 U / μL T7 reverse transcriptase, and RNase-free H2O.

[0019] In one particular embodiment, the reagent combination is used with 0.5 μL of the sample to be tested.

[0020] Furthermore, the reagent combination also includes substances for detecting other tuberculosis-specific markers.

[0021] A second objective of this invention is to provide the application of the aforementioned reagent combination in the detection of live Mycobacterium tuberculosis.

[0022] Furthermore, the application includes the following steps: S1. RNA extraction: Extracting RNA from the sample to be tested; S2, T7 reverse transcription reaction: The T7 promoter primer pair described in a) is used to perform a reverse transcription reaction to convert RNA into cDNA, which is then transcribed back into RNA by the T7 reverse transcriptase described in a). S3, CRISPR-Cas13a reaction: The reverse transcription product was further reacted in the CRISPR-Cas13a reaction system at 37°C for 30 minutes; S4. Fluorescence signal detection: Fluorescence detection at 590nm. The activity of Mycobacterium tuberculosis is determined by the presence and intensity of the fluorescence signal. If red fluorescence appears, the sample contains active Mycobacterium tuberculosis. If there is no red fluorescence, the sample contains no active Mycobacterium tuberculosis or no Mycobacterium tuberculosis.

[0023] Furthermore, the sample to be tested mentioned in S1 is selected from clinically collected samples, specifically sputum, saliva, and blood.

[0024] Furthermore, the fluorescence signal detection described in S4 is performed using a fluorescence microplate reader or a blue light transilluminator.

[0025] The beneficial effects of the technical solution of this invention are as follows: High specificity: Specific crRNAs are designed for the EF-Tu mRNA unique to live Mycobacterium tuberculosis. Only samples containing EF-TumRNA can produce significant fluorescent signals, while other pathogen samples do not show fluorescent signals, thus accurately identifying live bacteria and avoiding false positive results.

[0026] High sensitivity: The signal is amplified by T7 reverse transcription and CRISPR technology, and the detection sensitivity can reach 10 copies, which is suitable for early infection and low-load samples.

[0027] Rapid detection: By comparing experimental results at different temperatures and reaction times, the optimal reaction conditions (37℃, about 30 minutes) were determined. The entire detection process takes no more than 30 minutes, making it suitable for rapid diagnosis.

[0028] Visualized detection: Results are intuitively determined through fluorescence signals, requiring no complex equipment. Suitable for rapid on-site testing in primary healthcare units, it provides a basis for early treatment. It can also serve as a research tool for tuberculosis-related studies, such as drug screening and drug resistance research. Low cost: The testing is inexpensive and easy to operate, making it suitable for large-scale clinical applications. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the process for detecting Mycobacterium tuberculosis activity based on T7-CRISPR / Cas13a.

[0030] Figure 2 To validate the specificity of the T7-CRISPR / Cas13a-based method for detecting Mycobacterium tuberculosis activity.

[0031] Figure 3 To validate the sensitivity of the T7-CRISPR / Cas13a-based method for detecting Mycobacterium tuberculosis activity. Detailed Implementation

[0032] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0033] Example 1: T7-CRISPR-based Mycobacterium tuberculosis activity detection system reaction I. Design and synthesis of primers containing the target T7 promoter The tuf gene sequence of Mycobacterium tuberculosis was obtained from GenBank. A set of primers was designed based on the primer sites selected from the sequence. The primers containing the T7 promoter are: T7-1F: 5'-TAATACGACTCACTATAGGTGACACTGCCGGAGGGCACCGAGATGGTGA-3' (SEQ IDNO.1) T7-1R: 5'-TAATACGACTCACTATAGTGATGATCTTGGTGACCCGGCCGGCGCCCAC-3' (SEQ IDNO.2) T7-2F: 5'-TAATACGACTCACTATAGGGCACCGAGATGGTGATGCCCGGTGACAACA-3' (SEQ IDNO.5) T7-2R: 5'-TAATACGACTCACTATAGCTTGATGATCTTGGTGACCCGGCCGGCGCCC-3' (SEQ IDNO.6) T7-3F: 5'-TAATACGACTCACTATAGCACCGACGTGACCGGTGTGTGTGACACTGCCG-3' (SEQ IDNO.7) T7-3R: 5'-TAATACGACTCACTATAGCTACTTGATGATCTTGGTGACCCCGGCCGGCG-3' (SEQ IDNO.8) The T7 reverse transcription reaction reagent includes the aforementioned T7 promoter primers and T7 reverse transcriptase. The system contains 1 μL of 10× reverse transcription reaction buffer, with a final concentration of 1×; 0.5 μL of 50 U / μL T7 reverse transcriptase, with a total enzyme activity of 25 U per single reaction, resulting in a final concentration of 2.5 U / μL; 0.25 μL each of the 10 μM T7 promoter reverse transcription primer pair (the upstream primers are the nucleotide sequences shown in SEQ ID NO. 1, 3, and 5, and the downstream primers are the nucleotide sequences shown in SEQ ID NO. 2, 4, and 6), for a total sample volume of 1.5 μL, with a final concentration of 0.25 μM for each primer; 2 μL of RNA template from the sample to be tested; and the remaining amount is brought to 10 μL with RNase-Free H2O.

[0034] The system contained 2 μL of 10×Cas13a reaction buffer (final concentration 1×); 0.5 μL of 200 μM LwCas13a protein (final concentration 5 μM); 0.5 μL of 200 μM Mycobacterium tuberculosis-specific crRNA (SEQ ID NO.3) (final concentration 5 μM); 3 μL of 100 μM fluorescently labeled ssRNA reporter (SEQ ID NO.4) (final concentration 15 μM); 2 μL of the above T7 reverse transcription amplification product; and the remainder was brought to 20 μL with RNase-free H2O.

[0035] II. CRISPR / Cas13a Reaction System Prepare the reaction system. The premixed reaction system includes 2 μL of 10× reaction buffer, 0.5 μL of 200 μM Cas13a protein, 0.5 μL of 200 μM crRNA (SEQ ID NO.3), 3 μL of 100 μM ssRNA reporter (SEQ ID NO.4), 1 μL of 50 U / μL T7 reverse transcriptase, RNase-free H2O, and 0.5 μL of RNA from the sample to be tested.

[0036] III. Testing Steps S1. RNA extraction: Extracting RNA from the sample to be tested; The sample to be tested in this embodiment is: 1. Take 1 ml of clinical patient sample and place it in a 2 ml centrifuge tube. Add PBS buffer, homogenize thoroughly, and centrifuge at 3000 rpm for 5 min. Take the supernatant and repeat three times.

[0037] 2. Centrifuge at 14000 rpm for 5 minutes and collect the precipitate.

[0038] 3. Wash the precipitate three times with PBS (mix by pipetting and centrifugation).

[0039] 4. Resuspend the bacterial pellet in 100 μl of pre-cooled solution 1, mix thoroughly by pipetting and shaking vigorously to ensure even dispersion of the bacteria. Open the cap and let stand at room temperature for 3-5 minutes.

[0040] Solution 1: 50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0). Add 12.5 ml of 1 M Tris-HCl (pH 8.0), 10 ml of 0.5 M EDTA (pH 8.0), and 4.730 g of glucose to a final volume of ddH2O (500 ml). Autoclave for 15 min and store at 4°C.

[0041] 5. Add 200 μL of freshly prepared solution 2, tighten the cap, and invert 3-5 times to mix thoroughly. Perform the process gently. After lysing the cells and denaturing the DNA, place the centrifuge tube on ice for 5 minutes (to break hydrogen bonds). (Solution 2 is the lysis buffer, so the bacterial culture in the centrifuge tube will gradually become clear).

[0042] Solution 2: 0ml 10% SDS + 50ml NaOH + 400ml double-distilled water.

[0043] 6. Add 150 μl of pre-cooled solution 3, gently invert the tube 5-10 times to mix, and allow the DNA to renature. A white flocculent precipitate will appear. Place on ice for 5 minutes. Centrifuge at 12000 rpm for 10 minutes. The renaturation time should not be too long, otherwise genomic DNA contamination may occur.

[0044] Solution 3 is a neutralization solution. At this time, the plasmid DNA renatures, and the chromosomes and proteins undergo irreversible denaturation, forming an insoluble complex. At the same time, K+ causes the SDS-protein complex to precipitate.

[0045] Adjust the ratio of the three solutions according to the amount of bacteria to ensure that the volume ratio of solution 1: solution 2: solution 3 is 1:2:1.5.

[0046] 7. Transfer the supernatant into a clean EP tube, add 450 μl of one of phenol / chloroform / isoamyl alcohol, vortex to mix, and centrifuge at 12000 rpm for 5 min at 4 °C.

[0047] 8. Transfer the supernatant to another clean EP tube, add 2.5 times the volume of pre-cooled anhydrous ethanol and mix gently. Place in a -20°C refrigerator for 20 min, and centrifuge at 12,000 rpm for 10 min at 4°C.

[0048] 9. Discard the supernatant, wash the precipitate 1-2 times with 1 ml of pre-cooled 70% ethanol, centrifuge at 8000 rpm for 5 min at 4℃, discard the supernatant, and invert the EP tube onto filter paper to drain the water. Let it stand to allow the ethanol to evaporate completely.

[0049] 10. Add 20 μL of double-distilled water to the precipitate to dissolve the RNA, and store at -80°C for later use.

[0050] S2, T7 reverse transcription reaction: The T7 promoter primer pair of Example 1 was used to perform reverse transcription reaction to convert RNA into cDNA, which was then transcribed back into RNA by T7 reverse transcriptase; S3, CRISPR-Cas13a reaction: The reverse transcription product of S2 was added to the CRISPR-Cas13a reaction system of Example 2, and reacted at 37°C for 30 minutes. Figure 1 ).

[0051] IV. Result Interpretation The type of sample to be tested is determined by observing the fluorescence of CAL Fluor® Red 610. The activity of the tuberculosis sample is determined by observing the fluorescence of CAL Fluor® Red 610. If it is red and there is a significant difference compared with the negative control, it is judged to be a specific amplification product of the EF-Tu mRNA gene of Mycobacterium tuberculosis, and the sample is active Mycobacterium tuberculosis. If it does not glow and the fluorescence value is not significantly different from the negative control, it is judged that the T7 reverse transcription product does not contain the specific amplification product of the EF-Tu mRNA gene of Mycobacterium tuberculosis, and the sample is not Mycobacterium tuberculosis or is a dormant or dead Mycobacterium tuberculosis.

[0052] The negative control is a template-free negative control, which replaces the test sample with an equal volume of RNase-Free H2O. All other reagent components, dosages, and batches are identical to the test sample detection system. Example 2 Specificity Using gene sequences of other pathogenic microorganisms of different types synthesized by the company (as shown in the table below) and standard Mycobacterium tuberculosis EF-Tu mRNA containing EF-Tu mRNA (NC_000962.3, locus tag Rv0685, genomic location: complementary strand 784824→786014), the above samples were sequentially tested according to the T7-CRISPR-based activity detection system and procedure of Example 1 of this invention.

[0053]

[0054] The results are as follows: the fluorescence signal of the standard Mycobacterium tuberculosis sample remained basically unchanged before and after the reaction, similar to the negative control. However, the fluorescence signal of the Mycobacterium tuberculosis sample containing EF-Tu mRNA was significantly higher than that of the negative control after the reaction (P<0.01). For heat-inactivated Mycobacterium tuberculosis bacteria, H1N1 influenza A virus, Klebsiella pneumoniae, human bocavirus, adenovirus, and other non-tuberculous pathogenic microorganism samples, the fluorescence signals after the reaction were not significantly different from the negative control (P>0.05), and no positive fluorescence signals were detected. These results indicate that the detection method of the Mycobacterium tuberculosis activity detection system based on T7-CRISPR / Cas and EF-Tu mRNA constructed in this invention has good specificity. Figure 2 ).

[0055] Example 3 Sensitivity To determine the sensitivity of a T7-CRISPR-based tuberculosis viability detection system, the standard EF-TumRNA gene sequence was diluted to 10-1. 4 Copy / μl, 10 3 Copy / μl, 102 Copy / μl, 10 1 Six concentration gradients of 1 copy / μl, 1 copy / μl, and 0.1 copy / μl were used as templates, and detection was performed according to the detection system in Example 1.

[0056] The results are as follows: 10 1 ~10 4 The fluorescence signal after amplification of 10 copies / μl template for 30 min was significantly higher than that of the negative control. These results indicate that the EF-Tu mRNA gene detection system for Mycobacterium tuberculosis based on the T7-CRISPR system established in this invention can detect 10 copies of the sample and has high sensitivity. Figure 3 ).

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A reagent combination for detecting live Mycobacterium tuberculosis, characterized in that, The reagent combination includes: a) T7 reverse transcription reagent; b) CRISPR-Cas13a reaction system; a) The T7 reverse transcription reaction reagent includes a T7 promoter primer and a T7 reverse transcriptase; The T7 promoter primer is: T7-1F: 5'-TAATACGACTCACTATAGGTGACACTGCCGGAGGGCACCGAGATGGTGA-3'; T7-1R: 5'-TAATACGACTCACTATAGTGATGATCTTGGTGACCCGGCCGGCGCCCAC-3'; T7-2F: 5'-TAATACGACTCACTATAGGGCACCGAGATGGTGATGCCCGGTGACAACA-3'; T7-2R: 5'-TAATACGACTCACTATAGCTTGATGATCTTGGTGACCCGGCCGGCGCCC-3'; T7-3F: 5'-TAATACGACTCACTATAGCACCGACGTGACCGGTGTGGTGACACTGCCG-3'; T7-3R: 5'-TAATACGACTCACTATAGCTACTTGATGATCTTGGTGACCCCGGCCGGCG-3'; b) The CRISPR-Cas13a reaction system includes specific crRNA and fluorescently labeled ssRNA reporter: The specific crRNA has the nucleotide sequence shown in SEQ ID NO.3: 5'-uaauuucuacuaaguguagauGCGAUCCGCGAGGGUGGCCGCAC-3; The fluorescently labeled ssRNA reporter has the nucleotide sequence shown in SEQ ID NO.4: 5'-CAL Fluor® Red 610-UUUUUU-(Int BHQ2 dT)-3'.

2. The reagent combination according to claim 1, characterized in that, b) The CRISPR-Cas13a reaction system also includes a reaction buffer, Cas13a protein, RNase-Free H2O, and T7 reverse transcriptase.

3. The reagent combination according to claim 2, characterized in that, The CRISPR-Cas13a reaction system contains: 2 μL 10× reaction buffer, 0.5 μL 200 μM Cas13a protein, 0.5 μL 200 μM crRNA, 3 μL 100 μM ssRNA reporter, 1 μL 50 U / μL T7 reverse transcriptase, and RNase-free H2O.

4. The reagent combination according to claim 1, characterized in that, The reagent combination also includes substances for detecting other tuberculosis-specific markers.

5. The use of the reagent combination according to any one of claims 1 to 4 in the detection of live Mycobacterium tuberculosis.

6. The application according to claim 5, characterized in that, The application includes the following steps: S1. RNA extraction: Extracting RNA from the sample to be tested; S2, T7 reverse transcription reaction: The T7 promoter primer pair described in a) is used to perform a reverse transcription reaction to convert RNA into cDNA, which is then transcribed back into RNA by the T7 reverse transcriptase described in a). S3, CRISPR-Cas13a reaction: Add the reverse transcription product to the CRISPR-Cas13a reaction system described in b), and react at 37°C for 30 minutes; S4. Fluorescence Signal Detection: Fluorescence detection at 590nm. The activity of Mycobacterium tuberculosis is determined by the presence and intensity of the fluorescence signal. If red fluorescence is observed and there is a significant difference compared to the negative control, it is determined to be a specific amplification product of the EF-Tu mRNA gene of Mycobacterium tuberculosis, indicating that the sample is an active Mycobacterium tuberculosis. If no fluorescence is observed and the fluorescence value is not significantly different from the negative control, it is determined that the T7 reverse transcription product does not contain a specific amplification product of the EF-Tu mRNA gene of Mycobacterium tuberculosis. The negative control is a template-free negative control, which replaces the test sample with an equal volume of RNase-Free H2O, and is otherwise completely consistent with the test sample detection system.

7. The application according to claim 6, characterized in that, The sample to be tested in S1 is selected from clinically collected samples, which are selected from sputum, saliva, and blood.

8. The application according to claim 6, characterized in that, The fluorescence signal detection described in S4 is performed using a fluorescence microplate reader or a blue light transilluminator.

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