CRISPR (clustered regularly interspaced short palindromic repeats) system and kit for detecting drug-resistant tuberculosis

Abstract

The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats) system and a kit for detecting drug-resistant tuberculosis, and belongs to the technical field of drug-resistant tuberculosis detection. The CRISPR system comprises AaCas12b and sgRNA; the sgRNA comprises a first sgRNA of a targeting tuberculosis specific sequence IS6110, and a second sgRNA of a targeting tuberculosis specific sequence IS6110; the kit further comprises a second sgRNA and a third sgRNA which are used for targeting drug-resistant tuberculosis rpoBS531L and / or katGS315T mutation sites. On the basis of a Crispr-AaCas12b system, a tuberculosis specific sequence IS6110 and the most common mutation site of drug-resistant tuberculosis are targeted, and the Crispr-AaCas12b can specifically recognize the drug-resistant mutation site and does not recognize a wild type sequence by selecting PAM (Polyacrylamide), adjusting the length of an sgRNA spacer binding region and artificially introducing base mismatch; and the drug-resistant tuberculosis is rapidly and accurately detected through fluorescence signal monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug-resistant tuberculosis detection, and in particular relates to a CRISPR system, a kit and applications thereof for detecting drug-resistant tuberculosis. Background Art

[0002] According to the World Health Organization (WHO), the prevalence of drug-resistant tuberculosis remains severe worldwide. The prevalence of rifampicin-resistant tuberculosis (RR-TB), isoniazid-resistant tuberculosis (HR-TB) and multidrug-resistant tuberculosis (MDR-TB) not only increases the difficulty of treatment, but also increases the economic burden on patients and social public health pressure, posing a major challenge to global tuberculosis control and a huge obstacle to achieving the global goal of ending the tuberculosis epidemic by 2035. Rifampicin and isoniazid are the most powerful first-line anti-tuberculosis drugs. Resistance to either of them will increase the risk of tuberculosis treatment failure, relapse or resistance to other drugs. Therefore, being able to distinguish between common tuberculosis and drug-resistant tuberculosis in a timely and accurate manner is of great significance for the correct treatment and effective control of the disease.

[0003] At present, the diagnosis of drug-resistant tuberculosis mainly relies on traditional drug sensitivity tests, which generally take about 4 to 6 weeks to obtain drug sensitivity results. Failure to distinguish drug-resistant tuberculosis in a timely manner will lead to delays in the treatment of drug-resistant tuberculosis patients. Molecular diagnosis provides a new means for the rapid diagnosis of drug-resistant tuberculosis; molecular biological detection methods are fast and accurate, which can shorten the diagnosis time of drug-resistant tuberculosis; in addition to efficient gene editing, the CRISPR-Cas system can also perform gene testing to achieve the purpose of diagnosis. However, although there are certain differences in the recognition of mutations and wild types in the current detection of drug-resistant tuberculosis, the CRISPR-Cas system also has accessory cutting activity for wild-type genes. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a CRISPR system and a kit for detecting drug-resistant tuberculosis. The present invention is based on the Crispr-AaCas12b system, targets the tuberculosis-specific sequence IS6110, and the most common rpoBS531L (1349T>T) and katGS315T (944G>C) mutation sites of drug-resistant tuberculosis, and establishes a rapid detection system and a kit for drug-resistant tuberculosis.

[0005] The present invention provides a CRISPR system for detecting drug-resistant tuberculosis, comprising AaCas12b and sgRNA; the sgRNA comprises a first sgRNA targeting a tuberculosis-specific sequence IS6110; and further comprises a second sgRNA targeting a rpoBS531L mutation site of rifampicin-resistant tuberculosis and / or a third sgRNA targeting a katGS315T mutation site of isoniazid-resistant tuberculosis.

[0006] Preferably, the DNA template sequence for transcribing the first sgRNA is as shown in SEQ ID NO.3.

[0007] Preferably, the DNA template sequence for transcribing the second sgRNA is as shown in SEQ ID NO.6.

[0008] Preferably, the DNA template sequence for transcribing the third sgRNA is as shown in SEQ ID NO.9.

[0009] Preferably, the system further comprises a fluorescence monitoring sequence, and both ends of the fluorescence monitoring sequence are respectively labeled with a fluorescence reporter group and a fluorescence quencher group.

[0010] Preferably, the fluorescent reporter group is FAM, and the fluorescent quencher group is BHQ1.

[0011] Preferably, when lateral flow chromatography is used for detection, one end of the fluorescence monitoring sequence is connected to a fluorescent reporter group, and the other end is connected to biotin.

[0012] Preferably, the fluorescence monitoring sequence is 5'-TTTTTTT-3'.

[0013] The present invention also provides a kit for detecting drug-resistant tuberculosis, comprising the CRISPR system.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Based on the Crispr-AaCas12b system, the present invention targets the tuberculosis-specific sequence IS6110, and the most common rpoBS531L (1349T>T) and katGS315T (944G>C) mutation sites of drug-resistant tuberculosis, and establishes a rapid detection system and kit for drug-resistant tuberculosis. The present invention adjusts the length of the sgRNA spacer binding region and artificially introduces base mismatches in the sgRNA spacer binding region, so that Crispr-AaCas12b can specifically identify drug-resistant mutation sites, does not recognize wild-type sequences, and does not cut wild-type. The CRISPR system and kit provided by the present invention can quickly and accurately detect drug-resistant tuberculosis through fluorescent signals. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the recognition of tuberculosis-specific sequence IS6110;

[0016] Figure 2 Schematic diagram of detection of rpoBS531L mutation in rifampicin-resistant tuberculosis;

[0017] Figure 3 Schematic diagram of the detection of katGS315T mutation in isoniazid-resistant tuberculosis;

[0018] Figure 4 Screening of sgRNAs for detecting wild-type and mutant rpoB; A (left): sgRNAs with different lengths of spacer regions detect rpoB wild-type plasmid; (right): sgRNAs with different lengths of spacer regions detect rpoBS531L (1349C>T) mutant plasmid, agarose gel electrophoresis; (B): a series of sgRNAs with artificially introduced IDM in the 20nt spacer region detect rpoB wild-type and mutant plasmids, agarose gel electrophoresis; (C): a series of sgRNAs with artificially introduced IDM in the 20nt spacer region detect rpoB wild-type and mutant plasmids, urea Page gel electrophoresis;

[0019] Figure 5 This is the detection result of rpoB wild-type and mutant plasmids;

[0020] Figure 6 Screening of sgRNA for detecting wild and mutant katG; A: The first group of sgRNA, PAM motif is TTC, the length of the spacer region is 22nt, artificially introduced mismatch, detection of katG wild and mutant plasmid results, agarose gel electrophoresis; B: The second group of sgRNA, PAM motif is TAC, different lengths of spacer regions, detection of katG wild and mutant plasmid results, agarose gel electrophoresis; C: The second group of sgRNA, PAM motif is TAC, different lengths of spacer regions, detection of katG wild and mutant plasmid results, fluorescence monitoring; D: The third group of sgRNA, PAM motif is TAC, the length of the spacer region is 19nt, artificially introduced mismatch, detection of katG wild and mutant plasmid results, agarose gel electrophoresis; E: The third group of sgRNA, PAM motif is TAC, the length of the spacer region is 19nt, artificially introduced mismatch, detection of katG wild and mutant plasmid results, urea Page gel electrophoresis;

[0021] Figure 7 This is the detection result of katG wild-type and mutant plasmids;

[0022] Figure 8 It is a schematic diagram of the detection process;

[0023] Fig. 9It is the fluorescence monitoring result of clinical samples; NC: no sample to be tested; PC-WT: H37Rv; PC-M: MDR-TB; 1-39 represents samples 1-39;

[0024] Fig.10 Testing lateral flow chromatography results for clinical samples;

[0025] Fig.11 The results are shown in Figure 2. DETAILED DESCRIPTION

[0026] The present invention provides a CRISPR system for detecting drug-resistant tuberculosis, comprising AaCas12b and sgRNA; the sgRNA comprises a first sgRNA targeting a tuberculosis-specific sequence IS6110; and further comprises a second sgRNA targeting a rpoBS531L mutation site of rifampicin-resistant tuberculosis and / or a third sgRNA targeting a katGS315T mutation site of isoniazid-resistant tuberculosis.

[0027] In the present invention, the forward and reverse sequences of the tuberculosis-specific sequence IS6110 are as follows:

[0028] Starting from base 161 (161nt):

[0029]

[0030] in PAM motif.

[0031] The DNA template sequence for transcribing the sgRNA targeting the tuberculosis-specific sequence IS6110 is shown in SEQ ID NO.3, and is as follows:

[0032]

[0033] In the present invention, when detecting rifampicin-resistant tuberculosis, the forward and reverse sequences of the targeted drug-resistant tuberculosis rpoBS531L are as follows:

[0034] Starting from 1331 nt

[0035]

[0036] Among them, TTG is the PAM motif.

[0037] The DNA template sequence for transcribing the second sgRNA is shown in SEQ ID NO.6, and is as follows:

[0038]

[0039] The italic mark is the spacer area. Mismatched bases were artificially introduced.

[0040] In the present invention, when detecting isoniazid-resistant tuberculosis, the forward and reverse sequences of the targeted drug-resistant tuberculosis katGS315T (944G>C) are as follows:

[0041] Starting from 931 nt

[0042]

[0043] in It is the PAM motif.

[0044] The DNA template sequence for transcribing the third sgRNA is shown in SEQ ID NO.9, and is as follows:

[0045]

[0046] The italic mark is the spacer area, Artificially introduce mismatched bases

[0047] In the present invention, the system further comprises a fluorescence monitoring sequence, the two ends of which are respectively labeled with a fluorescence reporter group and a fluorescence quencher group; the fluorescence reporter group is FAM, and the fluorescence quencher group is BHQ1; the fluorescence monitoring sequence is preferably 5'-TTTTTTT-3' (SEQ ID NO.10).

[0048] The present invention also provides a kit for detecting drug-resistant tuberculosis, comprising the CRISPR system.

[0049] In the present invention, the test sample of the kit includes sputum or alveolar lavage fluid.

[0050] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] Example 1 Design and screening of sgRNA

[0052] Tuberculosis Drug Resistance Mutation Database ( DRAG ) and TheComprehensiveAntibiotic Resistance Database( TheComprehensiveAntibioticResist anceDatabase), including 78 rifampicin resistance-related studies and 98 isoniazid resistance-related studies. The data showed that the most common codon mutation in the rpoB gene was Ser531Leu (S531L), with a genotype mutation of 1349C>T, accounting for about 55.46% (5496 / 9910) of all rpoB gene mutations in rifampicin-resistant tuberculosis. The most common codon mutation in the katG gene was Ser315Thr (S315T), with a genotype mutation of 944G>C, accounting for about 62.03% (1617 / 2607) of all katG gene mutations in isoniazid-resistant tuberculosis.

[0053] The AaCas12b nuclease approaches the target DNA through the guidance of sgRNA, and when the sgRNA sequence is complementary to the base pair of the spacer region after the PAM motif of the target DNA sequence, AaCas12b can exert nuclease activity and has trans-cutting activity, that is, accessory cutting activity. AaCas12b also has high fidelity, high activity at room temperature, and is active under a wide range of temperature conditions. Therefore, AaCas12b is selected for in vitro diagnosis.

[0054] 1. Tuberculosis diagnosis: tuberculosis-specific sequence IS6110

[0055] Starting from 161 nt

[0056]

[0057] DNA template sequence for transcription of sgRNA

[0058]

[0059] 2. Rifampicin resistance test: rpoBS531L (1349C>T)

[0060] The PAM motif required by AaCas12b needs to contain T bases as much as possible, such as TTT, TTN, TNN, etc., and the more T bases it contains, the higher the activity and efficiency of the subsequent guided activation nuclease will be.

[0061] However, the genome of Mycobacterium tuberculosis has a high content of CG bases and a low content of AT bases. It is difficult to find a PAM motif containing more than one T base near the most common mutation site. By using the rpoBS531L (1349C>T) mutation, plus the original T base, it became the PAM motif TTG that can make the nuclease have higher activity.

[0062] rpoBS531L (1349C>T) starts from base 1331

[0063]

[0064] The backbone region of the DNA template sequence for transcribed sgRNA remains unchanged, as follows:

[0065]

[0066] The length of the spacer area is changed. Try the following lengths:

[0067]

[0068] The study found that when the sgRNA spacer binding region was 20 nt in length, AaCas12b had a higher efficiency in recognizing the rpoB 1349C>T mutation than the wild type. Figure 4 A in the sgRNA spacer. Although the difference between the two is statistically significant, the fluorescence signal monitoring showed that AaCas12b also exerted a low level of accessory cutting activity in the wild-type template system. Therefore, a pair of base mismatches (introduced double-base mutations, IDM) were artificially introduced at different positions in the sgRNA spacer binding region. When the rpoB 20nt gIDM, that is, the mutation formed a mismatch at the 13th and 14th positions in the spacer region after the PAM motif, the rpoB 1349C>T mutation could be effectively recognized, and the wild-type system nuclease was not activated. Figure 2 , Figure 4 B and C in Figure 5 .

[0069] The spacer area artificially introduces mismatches as follows:

[0070]

[0071]

[0072] 3. Isoniazid resistance test: katGS315T (944G>C)

[0073] For katGS315T (944G>C), the first group used TTC of the template chain near the mutation site as the PAM motif. By introducing a series of IDMs, there was no significant difference between the wild-type and mutant systems. Since no other adjacent multiple T bases were found near the mutation site, a PAM motif containing a T base was selected for subsequent sgRNA screening. The second group used TAC as the PAM motif. By changing the length of the spacer region, it was found that when it was 19nt, the difference in nuclease activity was statistically significant, but the wild-type system still had an obvious fluorescence signal. Therefore, a series of IDMs were further introduced. The dIDM, that is, when the mismatch occurred at 7nt and 8nt, there was a relatively ideal difference between the two. See Figure 3 , Figure 6 D and E in Figure 7 .

[0074] The first group of sgRNAs used TTC as PAM and a spacer region of 22 nt. The results showed that there was no significant difference between the wild type and mutant types. Figure 6 A in.

[0075] katGS315T(944G>C)931nt:

[0076]

[0077] The DNA template sequence for transcribing sgRNA, with the backbone region sequence unchanged, is shown in SEQ ID NO.11;

[0078] The spacer region is 22 nt long, and mismatches are artificially introduced at different positions, as follows:

[0079]

[0080] The second group of sgRNAs used TAC containing a single T base as PAM and designed spacer regions of different lengths. When the spacer region length is 19 nt, The difference in nuclease activity was statistically significant, but the wild-type system still had obvious fluorescence signals, see Figure 6 B and C in.

[0081] The DNA template sequence for transcribing sgRNA, with the backbone region sequence unchanged, is shown in SEQ ID NO.11;

[0082] The length of the spacer region is changed, and a base mismatch is artificially introduced at a fixed position. The bold mark is the original mutant base of the katGS315T (944G>C) mutant type, and the underline mark is the artificially introduced mismatch, as follows:

[0083]

[0084] The third group of sgRNAs were designed based on sgRNAs with a spacer length of 19 nt, and mismatched IDMs were artificially introduced at different positions in the spacer region. The results showed katG 19ntdIDM, that is, when the mismatches artificially introduced in the spacer region of sgRNA are at positions 7, 8, and 13, it can effectively recognize the mutant katGS315T (944G>C) and does not recognize the wild type katG, see Figure 3 .

[0085] The DNA template sequence for transcribing sgRNA, with the backbone region sequence unchanged, is shown in SEQ ID NO.11;

[0086] Based on the second group of 19nt, base mismatches were artificially introduced at different positions in the spacer region. The bold marks are the original mutant bases of the katGS315T (944G>C) mutant type, and the underline marks are the artificially introduced mismatches.

[0087]

[0088] Example 2 Application of CRISPR system for detecting drug-resistant tuberculosis

[0089] Material:

[0090] 1. Nucleic acid extraction reagent: 2% sodium hydroxide (NaOH), 1.45% sodium citrate (SOD CITR), 0.5% N-acetyl-L-cysteine ​​(NALC), 1X PBS buffer.

[0091] 2. Nucleic acid amplification reagents were purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd.

[0092] Template: 4 plasmids, all four plasmid vectors are Pu57, with different target genes, namely rpoB widetype, rpoB S531L (1349C>T), katG widetype, and katG S315T (944G>C).

[0093] Entrusted by Shanghai Shenggong Biotechnology Co., Ltd.

[0094] Primers: Synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0095] The specific sequences are shown in Table 1.

[0096] Table 1 Amplification primer sequences

[0097]

[0098] 3. sgRNA transcription:

[0099] Template: The scaffold single-stranded DNA fragment was synthesized by GenScript, and the corresponding single-stranded DNA fragment containing the spacer region was synthesized by Qingke Biotechnology.

[0100] Reagents: sgRNA Transcription Purification Kit

[0101] 4. Crispr reaction:

[0102] Reaction buffer: NEBbuffer r2.1

[0103] Enzyme: AaCas12b purified by ourselves, expressed protein (AaCas12b) Recombinant plasmid: commissioned by GenScript Biotech Co., Ltd. to synthesize

[0104] Template: amplification product from the previous step

[0105] 5. Probe: (Results presentation)

[0106] Fluorescence monitoring: sequence FAM 5'-TTTTTTT-3'BHQ1, commissioned by Shanghai Sangon Biotechnology Co., Ltd.

[0107] Lateral flow chromatography: sequence FAM 5'-TTTTTTT-3'Biotin, commissioned by Shanghai Biotechnology Co., Ltd. for synthesis;

[0108] Test strips, Cas12 / 13 special nucleic acid detection test strip JY0301.

[0109] instrument:

[0110] 1. Nucleic acid extraction: pipette, mixer, centrifuge, metal bath

[0111] 2. Nucleic acid amplification: BioRad PCR instrument

[0112] 3.Crispr reaction-fluorescence monitoring: Bio-Rad real-time fluorescence quantitative PCR instrument CFX96

[0113] 4.Crispr reaction-lateral flow chromatography: Bio-Rad PCR instrument

[0114] step:

[0115] 1. sgRNA transcription: follow the steps in the reagent instructions.

[0116] 2. Nucleic acid extraction: N-acetyl-L-cysteine-sodium hydroxide method (NALC-NaOH) and boiling method. Add an equal volume of NALC-NaOH mixture to 10 ml of sputum, mix by inversion for 20 seconds, and let stand at room temperature for 15 minutes; add 1X PBS to 50 ml, and centrifuge at 3000 rcf for 15 minutes. Discard the supernatant, resuspend the bacterial precipitate in 100 μl of sterile water, boil at 100℃ for 10 minutes; cool at 4℃ for 30 minutes; centrifuge at 12000 rpm, 4℃ for 10 minutes; transfer the supernatant to a clean centrifuge tube and store at -20℃.

[0117] 3. Nucleic acid amplification: multiplex isothermal amplification (multiplex-RPA). System: Buffer A 29.5μl, IS6110-RPA-F 1μl, IS6110-RPA-R 1μl, rpoB-RPA-F 2μl, rpoB-RPA-R 2μl, katG-RPA-F 2μl, katG-RPA-R 2μl, nucleic acid extraction product 2μl, ddH2O 6μl, Buffer B 2.5μl, mix well, incubate at 37℃ for 20min. Note: All primer concentrations are 10μM.

[0118] 4. Crispr reaction: The system contains 1X NEBbufferr2.1, AaCas12b 100nM, sgRNA 100nM, probe (ssDNA reporter) 500nM, add 2μl of the amplification product in the previous step, and incubate at 37℃ for 10min.

[0119] The whole reaction process is shown in the schematic diagram Figure 8 shown.

[0120] 5. Results:

[0121] Fluorescence monitoring: The third step, the Crispr reaction, is carried out in the Bio-Rad real-time fluorescence quantitative PCR instrument CFX96, while monitoring the changes in the fluorescence signal. Positive result judgment criteria: After 10 minutes of reaction, the fluorescence signal is greater than the mean + 3 times the standard deviation of the negative control group (>mean+3SD). The same sample is measured three times in parallel, and ≥ one of them is positive, which is considered positive.

[0122] Table 2 Test results of the method of the present invention and the standard drug sensitivity test method for samples 1 to 39

[0123]

[0124]

[0125] Note: MDR-TB is multidrug-resistant tuberculosis; RR-TB is riffampicin-resistant tuberculosis; Hr-TB is isoniazid-resistant TB; MABC is Mycobacterium abscessus complex; kansasii is Mycobacterium kansasii; gordonae is Mycobacterium gordonae; malmoense is Mycobacterium malmoense; N: no sequencing result.

[0126] The fluorescence monitoring results are as follows Fig. 9As shown, the sensitivity of the Crispr-AaCas12b IS6110 method for detecting tuberculosis is 97.14% and the specificity is 100%; the sensitivity of rpoBS531L for detecting rifampicin-resistant tuberculosis is 66.67% and the specificity is 100%, and the sensitivity of katGS315T for detecting isoniazid-resistant tuberculosis is 79.17% and the specificity is 90.91%. It can be seen that the method of the present invention has very good specificity. Since a few drug-resistant mutations are caused by other mutation sites, the sensitivity of drug-resistant detection is lower than that of tuberculosis detection (IS6110).

[0127] Lateral flow chromatography: After the third step, the Crispr reaction, is carried out in a Bio-Rad PCR instrument for 10 minutes, 5 μl of the product is diluted to 50 μl with ddH2O and dripped onto the sample end of the lateral flow chromatography test strip, and the results are observed after 5 minutes.

[0128] Lateral flow chromatography results Fig.10 As shown, from left to right, the first bar is the negative control; the second bar is the positive control; bars 3-5 are the results for Escherichia coli, IS6110, rpoB, and katG are all negative; bars 6-8 are the results for the MTB standard strain H37Rv, IS6110 is positive, rpoB is negative, and katG is negative; bars 9-11 are the results for RR-TB, IS6110 is positive, rpoB is positive, and katG is negative; bars 12-14 are the results for MDR-TB, IS6110, rpoB, and katG are all positive.

[0129] The electrophoresis results of multiple isothermal amplification products are as follows Fig.11 As shown, A is the result of non-optimized constant temperature amplification, lane 1 is the negative control, and there is no primer in the system; lane 2 is the result of IS6110 constant temperature amplification; lane 3 is the result of rpoB constant temperature amplification; lane 4 is the result of katG constant temperature amplification; lane 5 is the result of non-optimized multiple constant temperature amplification of three genes; B is the result of multiple constant temperature amplification after optimizing the primer volume and diluting the template 10 times, and the template amount in lane 1 is 1.5×10 8 CFU / ml; Lanes 2-10 are templates of the amplification system diluted 10-fold in sequence; Lane 11 is a negative control, there is no nucleic acid extraction template in the amplification system; Lane 12 is a negative control, there is no primer in the system. The non-optimized part in A means: regardless of single amplification or multiple amplification, the amount of primer (10μM) added is 2μl; the optimized part in B means: as described in the previous step, IS6110-RPA-F 1μl, IS6110-RPA-R 1μl, rpoB-RPA-F 2μl, rpoB-RPA-R2μl, katG-RPA-F 2μl, katG-RPA-R2μl.

[0130] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A CRISPR system for detecting drug-resistant tuberculosis, characterized in that: It includes AaCas12b and sgRNA; the sgRNA includes a first sgRNA targeting the tuberculosis-specific sequence IS6110; and also includes a second sgRNA targeting the rpoBS531L mutation site of rifampicin-resistant tuberculosis and / or a third sgRNA targeting the katGS315T mutation site of isoniazid-resistant tuberculosis.

2. The CRISPR system according to claim 1, characterized in that The DNA template sequence for transcribing the first sgRNA is shown in SEQ ID NO.

3.

3. The CRISPR system according to claim 1, characterized in that The DNA template sequence for transcribing the second sgRNA is shown in SEQ ID NO.

6.

4. The CRISPR system according to claim 1, characterized in that The DNA template sequence for transcribing the third sgRNA is shown in SEQ ID NO.

9.

5. The CRISPR system according to claim 1, characterized in that The system also includes a fluorescence monitoring sequence, and both ends of the fluorescence monitoring sequence are respectively labeled with a fluorescence reporter group and a fluorescence quencher group.

6. The CRISPR system according to claim 5, characterized in that The fluorescent reporter group is FAM, and the fluorescent quencher group is BHQ1.

7. The CRISPR system according to claim 5, characterized in that When lateral flow chromatography is used for detection, one end of the fluorescence monitoring sequence is connected to a fluorescent reporter group, and the other end is connected to biotin.

8. The CRISPR system according to claim 5 or 7, characterized in that The fluorescence monitoring sequence is 5'-TTTTTTT-3'.

9. A kit for detecting drug-resistant tuberculosis, characterized in that: A CRISPR system comprising any one of claims 1 to 7.

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