A CRISPR system and kit for detecting drug-resistant tuberculosis

Abstract

The present invention provides a CRISPR system and a kit for detecting drug-resistant tuberculosis, which belong to the technical field of drug-resistant tuberculosis detection. The CRISPR system includes AaCas12b and sgRNA; the sgRNA includes a first sgRNA targeting the tuberculosis-specific sequence IS6110; and also includes a second and a third sgRNA targeting the drug-resistant tuberculosis rpoBS531L and / or katGS315T mutation sites. The present invention is based on the Crispr-AaCas12b system, targeting the tuberculosis-specific sequence IS6110 and the most common mutation sites of drug-resistant tuberculosis. By selecting PAM and adjusting the length of the sgRNA spacer binding region and artificially introducing base mismatches, Crispr-AaCas12b can specifically recognize drug-resistant mutation sites and does not recognize wild-type sequences; drug-resistant tuberculosis is quickly and accurately detected by 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 global prevalence of drug-resistant tuberculosis (TB) remains severe. 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 the pressure on social public health. This poses a major challenge to global TB control and is a significant obstacle to achieving the global goal of ending the TB epidemic by 2035. Rifampicin and isoniazid are the most powerful first-line anti-TB drugs. Resistance to either increases the risk of TB treatment failure, relapse, or resistance to other drugs. Therefore, being able to promptly and accurately distinguish between common TB and drug-resistant TB is crucial for proper treatment and effective disease control.

[0003] Currently, the diagnosis of drug-resistant tuberculosis (DR-TB) relies primarily on traditional drug susceptibility testing, which typically takes approximately 4 to 6 weeks to obtain results. This inability to distinguish DR-TB in a timely manner can lead to delayed treatment for patients with DR-TB. Molecular diagnostics offer a new approach for the rapid diagnosis of DR-TB. Molecular biology detection methods are fast and accurate, shortening the time it takes to diagnose DR-TB. In addition to efficient gene editing, the CRISPR-Cas system can also perform genetic testing, thereby achieving diagnostic results. However, while the CRISPR-Cas system currently used for DR-TB detection exhibits some discrepancies in its ability to recognize mutant and wild-type DNA, it also exhibits additional cleavage activity against 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 kit for detecting drug-resistant tuberculosis. The present invention is based on the Crispr-AaCas12b system, targeting the tuberculosis-specific sequence IS6110, and the most common rpoBS531L (1349T>T) and katGS315T (944G>C) mutation sites of drug-resistant tuberculosis, to establish a rapid detection system and 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 the tuberculosis-specific sequence IS6110; and further comprises 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.

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

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

[0008] Preferably, the DNA template sequence for transcribing the third sgRNA is shown as 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: the present invention is based on the Crispr-AaCas12b system, targeting the tuberculosis-specific sequence IS6110, and the most common rpoBS531L (1349T>T) and katGS315T (944G>C) mutation sites of drug-resistant tuberculosis, to establish 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 recognize drug-resistant mutation sites, does not recognize wild-type sequences, and does not cut wild-type sequences. The CRISPR system and kit provided by the present invention can quickly and accurately detect drug-resistant tuberculosis by 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: sgRNAs with artificially introduced IDM in the 20nt spacer region detect rpoB wild-type and mutant plasmids, agarose gel electrophoresis; C: 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 sgRNAs for detecting wild-type and mutant katG; A: The first group of sgRNAs, with a PAM motif of TTC and a spacer region length of 22 nt, artificially introduced mismatches, detected katG wild-type and mutant plasmids, and agarose gel electrophoresis; B: The second group of sgRNAs, with a PAM motif of TAC and spacer regions of different lengths, detected katG wild-type and mutant plasmids, and agarose gel electrophoresis; C: The second group of sgRNAs, with a PAM motif of TAC and spacer regions of different lengths, detected katG wild-type and mutant plasmids, and fluorescence monitoring; D: The third group of sgRNAs, with a PAM motif of TAC and a spacer region length of 19 nt, artificially introduced mismatches, detected katG wild-type and mutant plasmids, and agarose gel electrophoresis; E: The third group of sgRNAs, with a PAM motif of TAC and a spacer region length of 19 nt, artificially introduced mismatches, detected katG wild-type and mutant plasmids, and urea Page gel electrophoresis;

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

[0022] Figure 8 Schematic diagram of the detection process;

[0023] Figure 9The fluorescence monitoring results of clinical samples; NC: no sample to be tested; PC-WT: H37Rv; PC-M: MDR-TB; 1-39 represent samples 1-39;

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

[0025] Figure 11 The results are for multiplex isothermal amplification. 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 the tuberculosis-specific sequence IS6110; and further comprises 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.

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

[0028] Starting from base 161 (nt):

[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 base 1331 (1331nt)

[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] Among them, the italic mark is the spacer area. Mismatched bases are 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 the 931st base (931nt)

[0042]

[0043] in 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 introduced 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 fluorescent reporter group and a fluorescent quencher group; the fluorescent reporter group is FAM, and the fluorescent 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 with reference to the embodiments, but they should not be construed as limiting the scope of protection 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), which included 78 studies on rifampicin resistance and 98 studies on isoniazid resistance. The data showed that the most common codon mutation in the rpoB gene was Ser531Leu (S531L), with a genotype of 1349C>T, accounting for approximately 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 of 944G>C, accounting for approximately 62.03% (1617 / 2607) of all katG gene mutations in isoniazid-resistant tuberculosis.

[0053] The AaCas12b nuclease is guided to the target DNA by a sgRNA. When the sgRNA sequence complements the base pairing of the spacer region following the PAM motif in the target DNA sequence, AaCas12b can exert its nuclease activity, exhibiting trans-cleavage activity, also known as accessory cleavage 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 diagnostics.

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

[0055] Starting from base 161 (161nt)

[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 Mycobacterium tuberculosis genome has a high CG content and a low AT content. It is difficult to find a PAM motif containing more than one T near the most common mutation sites. By using the rpoBS531L (1349C>T) mutation, the addition of a single T base resulted in a PAM motif, TTG, that enhances nuclease 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 is 20 nt in length, AaCas12b has a higher efficiency in recognizing the rpoB 1349C>T mutation than the wild type. Figure 4 Although the difference between the two is statistically significant, fluorescence signal monitoring shows that AaCas12b also exerts 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 forms a mismatch at the 13th and 14th positions in the spacer region after the PAM motif, it can effectively recognize the rpoB 1349C>T mutation, and the wild-type system nuclease is 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 the TTC of the template strand 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 a significant fluorescence signal. Therefore, a series of IDMs were further introduced. 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 the 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 was changed, and a base mismatch at a fixed position was artificially introduced. The bold mark is the original mutated base of the katGS315T (944G>C) mutant, and the underline mark is the artificially introduced mismatch, as shown below:

[0083]

[0084] The third group of sgRNAs was designed based on the sgRNA with a spacer length of 19 nt, and mismatched IDMs were artificially introduced at different positions in the spacer region. katG 19ntdIDM, that is, the mismatches artificially introduced into the spacer region of sgRNA at positions 7, 8, and 13, can effectively recognize the mutant katGS315T (944G>C) but not 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 mutated bases of the katGS315T (944G>C) mutant type, and the underline marks are the artificially introduced mismatches.

[0087]

[0088] Example 2 Application of the CRISPR System for Detecting Drug-Resistant Tuberculosis

[0089] Material:

[0090] 1. Nucleic acid extraction reagents: 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 Shenggong Bioengineering (Shanghai) 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: NEB buffer r2.1

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

[0104] Template: amplification product from the previous step

[0105] 5. Probe: (Result presentation)

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

[0107] Lateral flow chromatography: sequence FAM 5'-TTTTTTT-3'Biotin, commissioned by Sangon Biotech (Shanghai) 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 (NALC-NaOH) and Boiling Methods. Add an equal volume of NALC-NaOH mixture to 10 ml of sputum, mix thoroughly 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 pellet in 100 μl of sterile water, boil at 100°C for 10 minutes, cool at 4°C for 30 minutes, and centrifuge at 12000 rpm at 4°C for 10 minutes. Transfer the supernatant to a clean centrifuge tube and store at -20°C.

[0117] 3. Nucleic Acid Amplification: Multiplex RPA. System: Buffer A 29.5 μl, IS6110-RPA-F 1 μl, IS6110-RPA-R 1 μl, rpoB-RPA-F 2 μl, rpoB-RPA-R 2 μl, katG-RPA-F 2 μl, katG-RPA-R 2 μl, nucleic acid extraction product 2 μl, ddH2O 6 μl, Buffer B 2.5 μl. Mix thoroughly and incubate at 37°C for 20 min. Note: All primers are at 10 μM.

[0118] 4. Crispr reaction: The system contains 1X NEB buffer r2.1, AaCas12b 100nM, sgRNA 100nM, probe (ssDNA reporter) 500nM, add 2μl of the amplified product in the previous step, and incubate at 37°C 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 performed on a Bio-Rad CFX96 real-time fluorescence quantitative PCR instrument, while monitoring changes in the fluorescence signal. A positive result is determined by a fluorescence signal greater than the mean + 3 standard deviations of the negative control group (>mean + 3SD) at 10 minutes of reaction. The same sample is measured in triplicate, and if at least one of the three positive results is detected, the result 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 stands for multidrug-resistant tuberculosis; RR-TB stands for rifampicin-resistant tuberculosis; Hr-TB stands for isoniazid-resistant TB; MABC stands for Mycobacterium abscessus complex; kansasii stands for Mycobacterium kansasii; gordonae stands for Mycobacterium gordonae; malmoense stands for Mycobacterium malmoense; N: no sequencing result.

[0126] Fluorescence monitoring results are as follows Figure 9As shown, the Crispr-AaCas12b IS6110 method has a sensitivity of 97.14% and a specificity of 100% for detecting tuberculosis; rpoBS531L has a sensitivity of 66.67% and a specificity of 100% for detecting rifampicin-resistant tuberculosis; and katGS315T has a sensitivity of 79.17% and a specificity of 90.91% for detecting isoniazid-resistant tuberculosis. 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. The results are observed after 5 minutes.

[0128] Lateral flow chromatography results Figure 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 Figure 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 10-fold dilution of the template, and the template amount in lane 1 is 1.5×10 8 CFU / ml; Lanes 2-10 show the template in the amplification system diluted 10-fold in sequence; Lane 11 is a negative control, with no nucleic acid extraction template in the amplification system; Lane 12 is a negative control, with no primers in the system. Unoptimized in A refers to the addition of 2 μl of primers (10 μM) for both single and multiplex amplifications; Optimized in B refers to the addition of 1 μl of IS6110-RPA-F, 1 μl of IS6110-RPA-R, 2 μl of rpoB-RPA-F, 2 μl of rpoB-RPA-R, 2 μl of katG-RPA-F, and 2 μl of katG-RPA-R, as described in the previous step.

[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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A CRISPR system for detecting drug-resistant tuberculosis, characterized in that The invention comprises AaCas12b and sgRNA; the sgRNA comprises a first sgRNA targeting the tuberculosis-specific sequence IS6110; and further comprises 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; The DNA template sequence used to transcribe the first sgRNA is shown in SEQ ID NO. 3; The DNA template sequence used to transcribe the second sgRNA is shown in SEQ ID NO.6; The DNA template sequence used to transcribe the third sgRNA is shown in SEQ ID NO.

9.

2. The CRISPR system according to claim 1, wherein The system further comprises a probe, wherein both ends of the probe are respectively labeled with a fluorescent reporter group and a fluorescent quencher group.

3. The CRISPR system according to claim 2, wherein The fluorescent reporter group is FAM, and the fluorescent quencher group is BHQ1.

4. The CRISPR system according to claim 2, wherein When lateral flow chromatography is used for detection, one end of the probe is connected to a fluorescent reporter group, and the other end is connected to biotin.

5. The CRISPR system according to claim 2 or 4, characterized in that The probe is 5'-TTTTTTTT-3'.

6. A kit for detecting drug-resistant tuberculosis, characterized in that: Comprising the CRISPR system according to any one of claims 1 to 5.

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