Composition, kit and application thereof for detecting mycobacterium tuberculosis

Abstract

The present invention relates to the field of gene detection technology, and in particular to a composition, a kit and its application for detecting Mycobacterium tuberculosis. It comprises RAA primers for the conserved sequence of Mycobacterium tuberculosis and Mycobacterium tuberculosis-specific crRNA; the crRNA sequence comprises any one of SEQ ID NO:2 or SEQ ID NO:4. The present invention utilizes the specificity of crRNA to effectively ensure the accuracy of nucleic acid detection of bacterial characteristic sequences, and can determine whether MTB exists in the sample by fluorescence or test paper within 1 hour at 37°C. The present invention can detect MTB conveniently and quickly, and does not require complex equipment such as multiplex PCR instruments. Only a constant temperature device and a simple fluorescence detection device are needed to quickly obtain observable results, providing a lower cost, faster and more convenient detection method for the detection of MTB, which is of great significance for the early monitoring and prevention and control of MTB infection.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a composition and a kit for detecting Mycobacterium tuberculosis and applications thereof. Background Art

[0002] Mycobacterium tuberculosis (MTB) is a bacterium that can cause zoonotic diseases through aerosol transmission. According to the "Global Tuberculosis Report 2021", approximately 2 billion people worldwide are infected with Mycobacterium tuberculosis and 9.9 million people suffer from tuberculosis. India, China and Russia bear the heaviest burdens. As the second largest region in the world with multidrug-resistant tuberculosis, China has focused on developing new anti-tuberculosis drugs and studying the resistance mechanisms of Mycobacterium tuberculosis.

[0003] Due to the slow growth of Mycobacterium tuberculosis, the current traditional tuberculosis detection and drug resistance detection methods take up to several months, and relatively fast methods require complex instruments or imported reagents. The above factors have greatly hindered the effective prevention and control of tuberculosis in my country. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, which is a special family of DNA repeat sequences widely present in the genomes of bacteria and archaea. In recent years, the CRISPR / Cas system has been developed into a powerful tool for gene editing, which can perform precise editing of genes at a specific site. We used the CRISPR / Cas system to establish a specific detection method for Mycobacterium tuberculosis, and combined it with recombinase-mediated isothermal nucleic acid amplification technology (Recombinase Aided Amplification, RAA) to improve the sensitivity of the detection system and achieve "two-stage" amplification of the detection signal.

[0004] The number of registered tuberculosis patients worldwide in 2020 fell by 18% from 7.1 million to 5.8 million cases compared with 2019. In addition, the COVID-19 pandemic has also led to a 15% decrease in the number of multidrug-resistant / rifampicin-resistant tuberculosis patients receiving treatment, a 22% decrease in patients receiving preventive treatment, and a 9% decrease in the cost of tuberculosis prevention, diagnosis and treatment services. Due to the interruption of basic tuberculosis services, it is estimated that the number of tuberculosis deaths worldwide in 2020 increased by approximately 100,000 cases, so rapid, accurate and affordable Mycobacterium tuberculosis detection technology is urgently needed. Summary of the invention

[0005] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing MTB early monitoring and prevention technology, and to provide a combination and kit for detecting MTB with high sensitivity, high specificity, simple operation method, rapidity and accuracy, without the need for expensive instruments and equipment and professional standard laboratories, so as to realize the monitoring and prevention of MTB.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: a composition for detecting Mycobacterium tuberculosis, comprising RAA primers targeting conserved sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis-specific crRNA;

[0007] The crRNA sequence includes any one of SEQ ID NO: 2 or SEQ ID NO: 4.

[0008] Further, the primers include the sequences shown as SEQ ID NO:5 and SEQ ID NO:6, or the primers include the sequences shown as SEQ ID NO:7 and SEQ ID NO:8.

[0009] The present invention also includes a kit for detecting Mycobacterium tuberculosis, comprising the above-mentioned composition for detecting Mycobacterium tuberculosis.

[0010] Furthermore, it also includes Cas12a protein and a reporter single-stranded DNA molecular probe.

[0011] Furthermore, the Cas12a protein is LBCas12a; the reporter single-stranded DNA molecular probe sequence is: 5′-FAM-TTTTTT-BHQ1-3′.

[0012] Furthermore, it also includes RAA amplification system and CRISPR / Cas12a detection system reagents;

[0013] The present invention also includes the use of the above-mentioned composition for detecting Mycobacterium tuberculosis in developing and / or preparing products for tuberculosis diagnosis and / or prognosis assessment.

[0014] The present invention also includes a test strip for detecting Mycobacterium tuberculosis, which comprises the above-mentioned composition for detecting Mycobacterium tuberculosis and / or is constructed using the components in the above-mentioned kit.

[0015] The present invention also includes a method for detecting Mycobacterium tuberculosis for non-diagnostic and therapeutic purposes, comprising the following steps:

[0016] (1) taking a sample to be tested and extracting the MTB genome;

[0017] (2) RAA isothermal amplification: amplify the genome obtained in step (1) by the RAA method using the above primers;

[0018] (3) CRISPR / Cas12a detection: Take the amplified product in step (2), add the reporter single-stranded DNA molecular probe, Cas12a protein and the above-mentioned crRNA, perform CRISPR / Cas12a detection, and read the detection signal.

[0019] Furthermore, the method can distinguish 10 6 CFU / mL, 10 7 RAA amplification time of the CFU / mL MTB fluorescence value gradient.

[0020] The beneficial effects of the composition, kit and application thereof for detecting Mycobacterium tuberculosis of the present invention are:

[0021] The present invention screened out the best crRNA, which further improved the sensitivity of the method established by the present invention. The core of the CRISPR / Cas12a detection method lies in crRNA, so crRNA is directly related to sensitivity and accuracy. Among the four crRNAs, crRNA2 has the fastest reaction speed, the highest fluorescence value, the best effect, and the highest sensitivity through CRISPR / Cas12a detection.

[0022] The CRISPR / Cas system established in the present invention in combination with RAA isothermal amplification technology can realize rapid screening of pathogens. In addition to being able to use instruments such as ELISA instruments for accurate detection, it can also use test strips for auxiliary verification. It is easy to operate and can realize rapid detection of Mycobacterium tuberculosis, which has very broad market prospects and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the fluorescence value measurement results of 30 min of CRISPR / Cas12a detection of MTB by 4 crRNAs in Example 1 of the present invention;

[0024] Figure 2 This is a graph showing the fluorescence value measurement results of CRISPR / Cas12a detection of 7 different strains in an embodiment of the present invention;

[0025] Figure 3 The fluorescence value measurement results of CRISPR / Cas12a detection after RAA amplification for 30 minutes on samples with different concentrations in the embodiment of the present invention;

[0026] Figure 4 It is the fluorescence value measurement result of the optimal final concentration of Cas12a protein, crRNA and probe in the CRISPR / Cas12a detection system of the embodiment of the present invention;

[0027] Figure 5The present invention is for 2-15min RAA amplification time, for 10 6 CFU / mL, 10 7 The fluorescence value measurement results of CFU / mL MTB were detected by CRISPR / Cas12a system;

[0028] Figure 6 This is a result diagram of the test strip verification of RAA amplification products with different concentrations according to an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0030] Embodiment 1:

[0031] RAA primers targeting conserved sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis-specific crRNA:

[0032] The core of the CRISPR / Cas12a detection method lies in crRNA, so the quality of crRNA is directly related to the sensitivity and accuracy of the detection method. In order to screen for more efficient crRNA, two crRNAs were designed for the two conserved sequences of MTB, IS6110 and IS1081 (Table 1).

[0033] Table 1 crRNA (2, 4)

[0034]

[0035] Embodiment 2:

[0036] RAA primers targeting conserved sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis-specific crRNA:

[0037] The core of the CRISPR / Cas12a detection method lies in crRNA, so the quality of crRNA is directly related to the sensitivity and accuracy of the detection method. In order to screen for more efficient crRNA, two crRNAs were selected for the two conserved sequences of MTB, IS6110 and IS1081 (Table 2), and the primer sequences are shown in Table 3.

[0038] Table 2 crRNA (1, 3)

[0039]

[0040] Table 3 Primer sequences (5-8)

[0041]

[0042] Embodiment 3:

[0043] Establishment of RAA isothermal amplification and CRISPR / Cas12a method for detecting MTB

[0044] 1. Extraction of MTB genome

[0045] The Mtb genome was extracted by boiling lysis method.

[0046] 2. RAA isothermal amplification

[0047] Table 4 RAA amplification system

[0048]

[0049] 2.1 Add 41.5 μL A Buffer, 2.0 μL upstream primer (10 μM), and 2.0 μL downstream primer (10 μM) to the detection unit tube containing the reaction dry powder;

[0050] 2.2 Add 2.0 μL of the DNA sample to be tested obtained by step 1 into the detection unit tube;

[0051] 2.3 Add 2.5 μL of B Buffer to the detection unit tube cap, cover the tube cap, mix thoroughly by inverting 5-6 times, and centrifuge at low speed for 10 seconds (Note: Whether the "sufficient mixing" in this step will determine the repeatability of the experimental results);

[0052] 2.4 Place the detection unit tube in a 39°C constant temperature metal bath (or constant temperature water bath, constant temperature incubator, etc.) and incubate for 30 minutes.

[0053] 2.5 After the reaction is completed, take 5-10 μL of the reaction system for electrophoresis detection.

[0054] 3. RAA reaction conditions: The reaction system is 50 μL system, the reaction temperature is 39°C, and the constant temperature incubation is 30 minutes.

[0055] 4. Establishment of CRISPR / Cas12a method for detecting MTB

[0056] 4.1. Operation steps

[0057] 4.1.1 Take out Cas12a protein, crRNA, FQ probe, 10×C buffer, and H2O and slowly thaw them on ice;

[0058] 4.1.2 Take 2 μL of Cas12a protein, crRNA, FQ probe, and 10×C buffer respectively and add them to the system and mix thoroughly;

[0059] 4.1.3 Add 7 μL H2O to the above reagent mixture;

[0060] 4.1.4 Add 5 μL of RAA amplification product to make up the reaction system to 20 μL;

[0061] 4.1.5 Place the mixed sample into a fluorescent quantitative PCR instrument, react at 37°C, and monitor the fluorescent signal in real time (Note: The entire CRISPR reaction needs to be carried out in the dark and on ice to protect the activity of various components);

[0062] 4.2. CRISPR reaction conditions: The reaction system was 20 μL system, the reaction temperature was 37°C, the constant temperature amplification was 1 hour, and the fluorescence signal was collected every 1 minute.

[0063] 4.3. System and concentration

[0064] The CRISPR / Cas12a detection system is as follows:

[0065] Table 5 CRISPR / Cas12a detection system

[0066]

[0067] The present invention combines the CRISPR / Cas system with the RAA isothermal amplification technology "two-stage amplification" reaction for the detection of Mycobacterium tuberculosis. Through the coupling of RAA isothermal amplification and Cas12a protein, it is possible to achieve two-stage amplification of "sequence amplification" (completed by RAA) plus "enzymatic cascade" (completed by Cas enzyme), thereby surpassing the sensitivity of single-stage amplification such as qPCR. At the same time, due to the high efficiency of RAA isothermal amplification and CRISPR / Cas system, the detection time is shortened, and its ultra-high specificity and sensitivity make it possible to directly detect Mycobacterium tuberculosis.

[0068] Embodiment 4:

[0069] A kit for detecting Mycobacterium tuberculosis comprises RAA primers targeting conserved sequences of Mycobacterium tuberculosis, Mycobacterium tuberculosis-specific crRNA, Cas12a protein, a reporter single-stranded DNA molecular probe, and RAA amplification system and CRISPR / Cas12a detection system reagents.

[0070] The Cas12a protein is LBCas12a; the reporter single-stranded DNA molecular probe sequence is: 5′-FAM-TTTTTT-BHQ1-3′.

[0071] The RAA amplification system is shown in Table 4, and the CRISPR / Cas12a detection system is shown in Table 5.

[0072] The detection method of the kit is shown in Example 3.

[0073] When MTB nucleic acid forms a ternary complex with Cas12a and crRNA, the RuvC domain of Cas12a in the complex exercises DNase activity, cutting single-stranded DNA (ssDNA reporter) labeled with fluorescent signals, and detecting fluorescence to know whether the sample to be tested contains MTB nucleic acid. Based on this, the present invention constructs a kit for rapid and accurate detection of MTB based on CRISPR / Cas12a combined with RAA isothermal amplification, designs high-quality crRNA with the highest accuracy, highest efficiency and highest sensitivity, and ensures the MTB detection effect.

[0074] The present invention designs and manufactures simple detection kits and instruments for detection methods. In addition to using a real-time fluorescence quantitative PCR instrument for accurate quantification, the detection method established by the present invention can also design an APP that relies on a mobile phone camera, and at the same time develop a Mycobacterium tuberculosis detection kit to achieve portable rapid qualitative detection.

[0075] Embodiment 5:

[0076] A test strip for detecting Mycobacterium tuberculosis, comprising the composition for detecting Mycobacterium tuberculosis of Example 1 or 2 and / or constructed by using the components in the kit of Example 4.

[0077] Preparation of test strips: The test strips consist of five parts: sample suction area, FAM antibody binding area, biotin avidin binding area (quality control line, C line), FAM antibody capture area (detection line, T line), and absorption pad. After all pads are pretreated, they are assembled along the adhesive part of the nitrocellulose membrane at one time, overlapped by 2mm, and then cut into 0.4cm wide strips, dried at room temperature and stored for later use.

[0078] Test strips for rapid detection:

[0079] 1. Operation steps

[0080] 1.1 Take out the corresponding number of test strips according to the number of test samples and mark them on the absorbent pad. Each test strip can only be used for a single test of a single sample;

[0081] 1.2 After the CRISPR reaction is completed, open the reaction tube and insert the sample absorption area of ​​the test strip into the reaction tube. The liquid level must not exceed the uppermost end of the binding area. Wait until the judgment area is completely soaked (about 1-2 minutes. When the external ambient temperature is low, the water absorption speed will be reduced and the soaking time of the judgment area will be prolonged). After the quality control line (C line) is colored, the test strip can be taken out. Read the test results directly according to the color of the test strip;

[0082] 1.3 Observe the results within 10 minutes after the quality control line (C line) develops color. If it is later than 10 minutes, it will be considered invalid.

[0083] 1.4 Record the test results, seal the test strips and discard them in a safe place.

[0084] 2. The recommended system and concentration are shown in Table 5. The cleavage reaction can be carried out at a constant temperature of 37°C for 30 minutes.

[0085] The present invention establishes a low-cost paper chromatography test strip for rapid detection. Utilizing the trans-cleavage effect of Cas12a protein, a FAM and Biotin double-labeled probe (FB probe) is designed, and accurate test results can be obtained within 1 hour. More importantly, the test strip is low in cost, which provides the possibility for early warning of Mycobacterium tuberculosis infection in developing countries with high infection rates and economically underdeveloped areas.

[0086] Embodiment 6:

[0087] Screening out the most efficient crRNA

[0088] Nucleic acid was extracted from MTB by boiling lysis method. RAA amplification was then performed according to the RAA amplification method and conditions in Example 3. Finally, the reaction was performed according to the CRISPR / Cas12a detection method and conditions for MTB in Example 3. The crRNA in the reaction system used 4 crRNAs in Examples 1 and 2, respectively. The reaction time was 30 minutes, and the fluorescence value was read using a real-time fluorescence quantitative PCR instrument after the reaction was completed.

[0089] The results are as follows Figure 1 As shown: after 30 minutes of reaction time, IS6110-crRNA2 has the fastest reaction speed and a relatively high fluorescence value, indicating that among the four crRNAs, this one has the best effect.

[0090] Embodiment 7:

[0091] CRISPR / Cas12a detects MTB specificity

[0092] Experimental materials: Human Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium smegmatis, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa were purchased from Beijing Kezhan Biotechnology Co., Ltd.

[0093] The genomes of the above strains were extracted by boiling lysis method, and then RAA amplification was performed according to the RAA amplification method and conditions in Example 3. Finally, the reaction was carried out according to the CRISPR / Cas12a detection method and conditions of MTB in Example 3, and the fluorescence value was read by real-time fluorescence quantitative PCR instrument after the reaction.

[0094] The results are as follows Figure 2As shown: the fluorescence values ​​of Mycobacterium smegmatis, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa are extremely low, while the fluorescence values ​​of the two Mycobacterium tuberculosis strains are above 200, and are significantly different from the other five strains (P < 0.0001). The above results show that the method for detecting MTB constructed by the present invention has strong specificity and has no cross reaction with other nucleic acids.

[0095] Embodiment 8:

[0096] CRISPR / Cas12a sensitivity in detecting MTB

[0097] The strain was cultured to 10 7 CFU / mL, and then the strain was diluted ten million times to 10 0 CFU / mL, and 8 groups of bacterial solutions with different concentrations were obtained. The eight groups of bacterial solutions were respectively subjected to the water boiling method to extract the genome, and then RAA amplification was performed according to the RAA amplification method and conditions in Example 3. Finally, the reaction was carried out according to the CRISPR / Cas12a detection MTB method and conditions in Example 4, and the fluorescence value was read using a real-time fluorescence quantitative PCR instrument after the reaction was completed.

[0098] The results are as follows Figure 3 As shown in the figure, as the concentration increases tenfold, the fluorescence curve increases successively. The standard curve of fluorescence intensity relative to the concentration of the target sequence is made by measuring the fluorescence signal value, and the detection line of the CRISPR / Cas system for Mycobacterium tuberculosis is calculated to be 10 0 CFU / mL of Mycobacterium tuberculosis, showing extremely high sensitivity.

[0099] Embodiment 9:

[0100] Optimal final concentrations of components of the CRISPR / Cas detection system

[0101] The optimal final concentration of the three components in the CRISPR system (Cas12a protein, crRNA, FQ probe) was explored. 6 The strain genome of CFU / mL was then subjected to RAA amplification according to the RAA amplification method and conditions in Example 3. Finally, the reaction was carried out according to the CRISPR / Cas12a detection MTB method and conditions in Example 3, and the fluorescence value was read using a real-time fluorescence quantitative PCR instrument after the reaction was completed.

[0102] The results are as follows Figure 4 As shown: It can be seen that when the final concentration of Cas12a protein is 200nM, the final concentration of crRNA is 200nM, and the final concentration of FQ probe is 500nM, the obtained fluorescence signal value is the highest.

[0103] Embodiment 10:

[0104] Effects of different RAA amplification times on fluorescence values ​​detected by CRISPR / Cas system

[0105] Due to the high amplification efficiency of RAA, for high-concentration genome amplification, the CRISPR / Cas system can obtain an amplification product with a fluorescence value gradient that cannot be distinguished in 30 minutes, as described in Example 8. Figure 3 , 10 6 CFU / mL, 10 7 After 30 minutes of RAA amplification, the fluorescence values ​​measured by the CRISPR / Cas system could not be distinguished. 6 CFU / mL, 10 7 CFU

[0106] / mL of MTB were subjected to 2-15min RAA amplification. Finally, the reaction was carried out according to the CRISPR / Cas12a detection MTB method and conditions in Example 3, and the fluorescence value was read using a real-time fluorescence quantitative PCR instrument after the reaction was completed.

[0107] Figure 5 As shown, AH are the fluorescence detection results of RAA amplification for 2, 3, 4, 5, 6, 9, 12, and 15 minutes, respectively. When the RAA reaction was 2 minutes and 3 minutes, both were specifically amplified; when the RAA reaction was 4 minutes and 5 minutes, the two could be distinguished by the CRISPR / Cas system; when the RAA reaction was 6-15 minutes, both were specifically amplified with high efficiency, but the gradient could not be distinguished.

[0108] Embodiment 11:

[0109] Test strip verification

[0110] Auxiliary verification is performed using the test strip method. According to the operating steps in Example 5, the FAM-Biotin double-labeled probe is designed. The probe is cut by the trans-cutting effect of Cas12a, and flows through the FAM antibody binding area, the avidin binding area (quality control line), and the FAM antibody capture area (detection line) in sequence. If it is positive, the detection line appears red bands due to the capture of the cut FAM antibody; if it is negative, FAM and Biotin are not separated, and they stagnate together in the biotin avidin binding area, and will not reach the detection line, so the detection line has no red bands; if there is no color development of any band in the test strip, it is judged to be invalid.

[0111] The results are as follows Figure 6As shown: for different concentrations of target fragments, it can be seen that the test strips all show obvious detection lines, and the detection line of the negative control (NC) does not show color, indicating that the test strip method also has high specificity and sensitivity. The test strip test can obtain accurate results within 1 hour, and the operation is relatively simple. For some testing institutions with incomplete testing facilities, it is very helpful for the preliminary screening of Mycobacterium tuberculosis.

[0112] In the above embodiments, the primers were synthesized by Guangzhou Aiki Biotechnology Co., Ltd.; crRNA and reporter single-stranded DNA molecular probes were synthesized by Guangzhou Bolais Biotechnology Co., Ltd.; Cas12a protein was purchased from Guangzhou Bolais Biotechnology Co., Ltd.

[0113] The above embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0114] Statistical analysis of the above examples: All experiments were repeated at least 3 times independently, and the results were expressed as mean values ​​and standard errors, and analyzed using T-tests. All statistical analyses used P<0.0001 as the test standard for significant statistical differences, and the analysis software was GraphPad Prism 8.

[0115] In the preparation method of the present invention, the addition order of various materials and the specific reaction steps can be adjusted by those skilled in the art, which is not only suitable for small-scale preparation in laboratories, but also suitable for industrial large-scale production in chemical plants. In industrial batch production, the specific reaction parameters can be determined by those skilled in the art through experiments.

[0116] Unless otherwise specified, the reagents, materials, etc. used in the above examples can be obtained from commercial sources or synthesized from raw materials obtained from commercial sources.

[0117] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable ordinary technicians in the field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Weifang Medical College <120> Composition, kit and application thereof for detecting mycobacterium tuberculosis <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 41 <212> RNA <213> Artificial Sequence <400> 1 uaauuucuac uaaguguaga uaucagcucg gucuuguaua g 41 <210> 2 <211> 41 <212> RNA <213> Artificial Sequence <400> 2 uaauuucuac uaaguguaga uaaccagucg acccagcgcg c 41 <210> 3 <211> 41 <212> RNA <213> Artificial Sequence <400> 3 uaauuucuac uaaguguaga ugaccaggcg cuccauccgg c 41 <210> 4 <211> 41 <212> RNA <213> Artificial Sequence <400> 4 uaauuucuac uaaguguaga uccggacugg cugcugcagc g 41 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <400> 5 ggtcggaagc tcctatgaca atgcactagc c 31 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 ttgagcgtag taggcagcct cgagttcgac 30 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <400> 7 ccaagctgcg ccagggcagc tatttcccgg ac 32 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <400> 8 ttggccatga tcgacacttg cgacttgga 29

Claims

1. A composition for detecting Mycobacterium tuberculosis, characterized in that: including RAA primers targeting conserved sequences of Mycobacterium tuberculosis and crRNA specific to Mycobacterium tuberculosis; The crRNA consists of the sequence shown in SEQ ID NO: 2; The primers consist of the sequences shown in SEQ ID NO:5 and SEQ ID NO:

6.

2. A kit for detecting Mycobacterium tuberculosis, characterized in that: A composition for detecting Mycobacterium tuberculosis according to claim 1; It also includes Cas12a protein and reporter single-stranded DNA molecular probe; The Cas12a protein is LBCas12a; the reporter single-stranded DNA molecular probe sequence is: 5′-FAM-TTTTTT-BHQ1-3′; It also includes RAA amplification system and CRISPR / Cas12a detection system reagents.

3. Use of the composition for detecting Mycobacterium tuberculosis according to claim 1 in developing and / or preparing products for tuberculosis diagnosis and / or prognosis assessment.

4. A test strip for detecting Mycobacterium tuberculosis, characterized in that: The composition for detecting Mycobacterium tuberculosis according to claim 1 or the composition is constructed by using the components in the kit according to claim 2.

5. A method for detecting Mycobacterium tuberculosis for non-diagnostic and therapeutic purposes, characterized in that: The steps include: (1) Take the sample to be tested and extract the MTB genome; (2) RAA isothermal amplification: using the primers in claim 1 to amplify the genome obtained in step (1) by the RAA method; (3) CRISPR / Cas12a detection: Take the amplified product in step (2), add the reporter single-stranded DNA molecular probe, Cas12a protein and the crRNA in claim 1, perform CRISPR / Cas12a detection, and read the detection signal; The method can distinguish 10 6 CFU / mL, 10 7 RAA amplification time of the gradient of CFU / mL MTB fluorescence values.

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