crRNA, kit, and detection method for detecting mutations in the gyrA fluoroquinolone-resistance-related gene of Mycobacterium tuberculosis

Abstract

The application provides a crRNA, a kit and a detection method for detecting a gyrA fluoroquinolone drug resistance related gene mutation of mycobacterium tuberculosis. A spacer in the crRNA comprises a sequence shown in any one of SEQ ID No. 1 to SEQ ID No. 13. By introducing a base mismatch on the spacer in the crRNA molecule, high specificity and rapid detection of the gyrA gene fluoroquinolone drug resistance related mutation of mycobacterium tuberculosis are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to crRNA, a kit and a detection method for detecting gyrA fluoroquinolone drug resistance related gene mutation of Mycobacterium tuberculosis. BACKGROUND

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, which can invade many organs. Tuberculosis is one of the very serious infectious diseases, and is also a highly fatal infectious disease. According to the WHO estimate, there are about 1.7 billion people with latent tuberculosis infection in the world, accounting for about 1 / 4 of the total population. The new cases per year basically reach 80-100 million. The WHO reported in 2008 that the total drug resistance rate of tuberculosis in the world was 20.0%, and the multi-drug resistance rate was 5.3%, and it was estimated that there were 500,000 cases of multi-drug resistant tuberculosis in the world.

[0003] Quinolones are chemically synthesized antibacterial drugs. Quinolones target the DNA of bacteria, interfere with DNA gyrase, and further cause irreversible damage to bacterial DNA, achieving antibacterial effect. Fluorine-containing new quinolones, commonly known as fluoroquinolones, commonly used drugs include ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, sparfloxacin, gatifloxacin, etc. Fluoroquinolones show good activity against Mycobacterium tuberculosis, but the resistance of Mycobacterium tuberculosis to fluoroquinolones also gradually increases. Studies have shown that the mutation of the gyrA gene of Mycobacterium tuberculosis encoding DNA gyrase in the quinolone drug resistance determining region (QRDR) is the main mechanism of Mycobacterium tuberculosis producing fluoroquinolone resistance. The most common mutation sites related to fluoroquinolone drug resistance are GyrA 88, 90, 91 and 94.

[0004] The detection methods for Mycobacterium tuberculosis resistant to fluoroquinolone drug resistance related mutations include gene sequencing (DNA sequencing), real-time fluorescence quantitative PCR (real time PCR) and chip hybridization (DNA chip) and the like. The existing methods for detecting mutations require large instruments, are high in cost and time-consuming.

[0005] Zhang Feng and the Collinis group collaborated to report a high-sensitivity nucleic acid molecule detection toolkit based on CRISPR-Cas13a, which they called SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing). In addition to Cas13a and crRNA targeting the target to be detected, the system also includes an RNA probe that releases fluorescence after being cut. crRNA consists of a fixed direct repeat sequence and a target-specific spacer sequence. After Cas13a recognizes and cuts the target RNA under the guidance of crRNA, Cas13a exhibits non-specific RNase activity, exerting a side effect to continue cutting the RNA fluorescent probe and releasing a fluorescent signal. In the development of this tool, Zhang Feng's group also used an isothermal amplification method - recombinase polymerase amplification (Recombinase Polymerase Amplification) to amplify nucleic acids, which can make the detection limit of the tool reach the single-molecule level, with similar sensitivity to digital droplet PCR (ddPCR) and quantitative fluorescence PCR (qPCR), and with lower variability. In existing literature reports, the CRISPR-Cas13a system is mainly used to detect the presence of specific nucleic acid fragments, and the system has not yet been used to identify whether there are specific mutation sites in specific nucleic acid fragments.

[0006] Based on this, the present invention is proposed. Summary of the Invention

[0007] The present invention aims to provide a crRNA, kit, and method for detecting mutations in the gyrA gene of Mycobacterium tuberculosis associated with fluoroquinolone resistance. By introducing base mismatches into the designed crRNA molecule, highly specific and rapid detection of gyrA gene mutations in Mycobacterium tuberculosis is achieved. This method can be used to detect drug-resistant mutations in Mycobacterium tuberculosis caused by single-base mutations, such as fluoroquinolone-resistant mutations.

[0008] The technical solutions provided by the present invention are as follows:

[0009] A crRNA for detecting mutations in the fluoroquinolone-resistance-related gene gyrA of Mycobacterium tuberculosis, wherein the spacer sequence of the crRNA comprises a sequence shown in any one of SEQ ID No. 1 to SEQ ID No. 13.

[0010] The inventors proposed that by optimizing the number and position of mismatched bases in the spacer sequence in crRNA, crRNA can differentially guide Cas13a to recognize wild-type and mutant sequences, releasing significantly different RNA enzyme activities, and realizing the detection of single nucleotide mutations. They also used fluoroquinolone resistance-related mutations in Mycobacterium tuberculosis gyrA as detection targets and demonstrated a set of crRNAs that can distinguish wild-type sequences from mutant sequences.

[0011] In one embodiment, the crRNA is used to detect point mutations in the gyrA gene of Mycobacterium tuberculosis;

[0012] Preferably, the point mutation is a single base substitution resulting in a change in amino acids at positions 88, 90, 91, and 94 of gyrA.

[0013] A CRISPR-Cas13a system comprising any of the aforementioned crRNAs.

[0014] In one embodiment, the CRISPR-Cas13a system further comprises a plasmid capable of expressing the Cas13a protein, a signal reporter probe, and a nuclease buffer.

[0015] In one embodiment, the Cas13a protein is selected from LwCas13a or LshCas13a; preferably, the Cas13a protein is LwCas13a.

[0016] Application of the crRNA or the CRISPR-Cas13a system in detecting gyrA gene mutations in Mycobacterium tuberculosis;

[0017] Preferably, the application is for detecting fluoroquinolone-resistant mutations in Mycobacterium tuberculosis; the application is for non-disease diagnosis purposes.

[0018] A kit comprising the crRNA; preferably, the kit is used to detect fluoroquinolone-resistant mutations in Mycobacterium tuberculosis.

[0019] A method for detecting a Mycobacterium tuberculosis gyrA gene mutation for non-disease diagnosis purposes, the method comprising:

[0020] Obtaining RNA from the sample to be tested;

[0021] The crRNA is incubated with the sample RNA, LwCas13a protein, a fluorescently labeled probe, and an RNase inhibitor in a nuclease buffer system for detection.

[0022] In one embodiment, after incubation in the nuclease buffer system, the sample detection zone of the colloidal gold test strip is added for detection or the fluorescence value is measured by a fluorescence detector after incubation of the system; preferably, the fluorescence is detected every 1-2 min on an enzyme-labeled instrument.

[0023] In one embodiment, the content of each component in the system is: LwCas13a 400-500 nM; crRNA 800-1000 nM; sample RNA 1600-2000 nM; fluorescently labeled probe 120-200 nM; preferably; LwCas13a 450 nM; crRNA 900 nM; sample RNA 1800 nM; fluorescently labeled probe 120 nM.

[0024] In one specific embodiment of the present application, the 5' end of the sequence of the fluorescently labeled probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. The fluorescently labeled probe is cleaved by the activated LwCas13a nonspecific RNase and releases a fluorescent signal.

[0025] The crRNA forms a single base mismatch with the mutant ssRNA of the gene gyrA and a double base mismatch with the wild type ssRNA of the target gene.

[0026] The crRNA recognizes the mutant gene target ssRNA it targets, the Cas13a protein binds to the corresponding ssRNA containing the mutation site under the guidance of the set crRNA, the LwCas13a undergoes conformational change, obtains nonspecific RNAse activity, the Cas13a protein with RNAse activity cleaves the fluorescently labeled probe, and releases a fluorescent signal; the crRNA set for specific mutations cannot guide the system to bind to the wild type ssRNA well, and the nonspecific RNAse activity of LwCas13a cannot be well excited, so that the fluorescence signal difference between the wild type ssRNA and the mutant ssRNA can be distinguished by the set crRNA guided system, the mutant and wild type sites can be distinguished, the detection of the mutation of the Mycobacterium tuberculosis gyrA gene, especially the mutation of Mycobacterium tuberculosis caused by a single base, such as fluoroquinolone resistance mutation, can be realized.

[0027] Beneficial effects:

[0028] (1) The present application provides a crRNA that can specifically target multiple drug-resistant gene mutation sites (amino acids 88, 90, 91, and 94) of Mycobacterium tuberculosis and activate the RNAse activity of the CRISPR-Cas13a system by design, construction, and screening, which can specifically detect drug-resistant gene mutation sites.

[0029] (2) The mutation detection of the mycobacterium tuberculosis fluoroquinolone resistance related site in the application does not need a sequencer or a fluorescence quantitative PCR instrument to complete, only needs to use a simple instrument such as a fluorescence microplate reader or a naked-eye observation method such as colloidal gold to judge whether the RNAse activity of Cas13a is activated, which can be suitable for subsequent point of care (POCT) detection methods.

[0030] (3) The application focuses on detecting the main mutation sites (90 and 94), and covers other common mutation sites, achieving the effect of relatively comprehensive detection.

[0031] (4) The method of the application has low detection cost, can be repeatedly detected, and the results can be directly analyzed (for example, by fluorescence reading), which is suitable for large-scale clinical application.

[0032] (5) The application improves the sensitivity and specificity of the detection of mycobacterium tuberculosis drug resistance mutations, and shortens the detection time. The detection sensitivity of the application is high (the minimum detection limit reaches 1x100 copies / μL), the detection speed is fast, and the corresponding mutations can be detected in a short time (when the reaction is carried out for 5 minutes, the fluorescence signal of the mutant gene is obviously higher than that of the wild type gene). BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0034] Figure 1 The result graph of Example 1 of the application: A detection of the mutation (G88A) at position 88 of the gyrA gene, 88 3-5 crRNA, 88 4-6 crRNA, 88 5-7 crRNA, 88 6-8 crRNA respectively detecting wild type target and 88 amino acid mutant target; B detection of the mutation (G88A) at position 88 of the gyrA gene, 88 3-5 crRNA, 88 4-6 crRNA, 88 5-7 crRNA, 88 6-8 crRNA respectively detecting the fluorescence signal ratio of mutant ssRNA target and wild type target; (n=3 technical replicates; bar represents mean±SD);

[0035] Figure 2Figure 2 shows the results of Example 2 of the present invention: A shows the detection of mutations at position 90 (A90V and A90G) in the gyrA gene, where A90V 3-5crRNA, A90V 4-6crRNA, A90G 3-5crRNA, and A90G4-6crRNA respectively detect the wild-type target and targets with amino acid mutations at position 90 (A90V and A90G); B shows the detection of mutations at position 90 (A90V and A90G) in the gyrA gene, where A90V3-5crRNA, A90V4-6crRNA, A90G 3-5crRNA, and A90G 4-6crRNA respectively detect the fluorescence signal ratios of mutant ssRNA targets to wild-type targets; (n=3 technical replicates; bar represents mean±SD);

[0036] Figure 3 Figure 3 shows the results of Example 3 of the present invention: A shows the detection of the gyrA gene mutation at position 91 (S91P), where 91 10-17 crRNAs detect the wild-type target and the target with the amino acid mutation at position 91 (S91P), respectively; B shows the fluorescence signal ratio of the mutant ssRNA target to the wild-type target detected by 91 10-17 crRNAs for the detection of the gyrA gene mutation at position 91 (S91P); (n=3 technical replicates; bar represents mean±SD);

[0037] Figure 4 Figure 4 shows the results of Example 4 of the present invention: A shows the detection of mutations at position 94 (D94N, D94H, D94T, D94G) in the gyrA gene, where D94N 10-16crRNA, D94H 10-16crRNA, D94T 10-16crRNA, and D94G 17-10crRNA detect wild-type targets and targets with amino acid mutations at position 94 (D94N, D94H, D94T, and D94G), respectively; B shows the detection of mutations at position 94 (D94N, D94H, D94T, and D94G) in the gyrA gene, where D94N 10-16crRNA, D94H 10-16crRNA, D94T 10-16crRNA, and D94G 17-10crRNA detect the fluorescence signal ratios of mutant ssRNA targets to wild-type targets, respectively; (n=3 technical replicates; bar represent mean ± SD);

[0038] Figure 5Figure 5 shows the results of Example 5 of the present invention: The crRNA spacer sequence in the Cas13a / crRNA+Target RNA experiment is completely complementary to the gyrA gene 88 amino acid mutant (G88A) sequence (spacer set to 28 nt, no additional base mismatch); the negative control refers to the experiment in which no crRNA, Cas13a, or ssRNA was added to the system; (n=3 technical replicates; bar represents mean±SD). DETAILED DESCRIPTION

[0039] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the examples are conventional methods, and the materials, reagents, etc. used in the examples are all commercially available unless otherwise specified.

[0041] Example 1

[0042] 1. Design of crRNA targeting the 88th amino acid mutation of the gyrA gene of Mycobacterium tuberculosis

[0043] (1) Screening principles:

[0044] The principle of designing the spacer sequence of the designed crRNA is to adjust the number and relative position of bases that do not match the wild-type ssRNA in the spacer sequence, so that the selected crRNA for a specific mutation cannot recognize the wild ssRNA or recognizes it at a low level, and efficiently recognizes the corresponding mutant ssRNA, thereby releasing the non-specific RNase activity of Cas13a.

[0045] (2) Selection of wizard sequence:

[0046] According to the site of mutation, the length of crRNA spacer sequence is 28 nucleotides (nt). G CG C C), 4 crRNA guide sequences targeting it were designed (SEQ ID No. 1 to SEQ ID No. 4). The spacer sequences and their target sequences are shown in the following table, where the bold underlined parts are mutation sites.

[0047]

[0048] Among them, the crRNA spacer is 28nt, the third base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the fifth base of the spacer with either the wild type or the mutant type (i.e., 88spacer3-5) can distinguish between wild-type and mutant target genes (discrimination ability see Figure 1 ).

[0049]

[0050] Among them, the crRNA spacer is 28nt, the 4th base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the 6th base of the spacer with the wild type and the mutant type (i.e., 88spacer4-6) can distinguish between wild-type and mutant target genes (discrimination ability see Figure 1 ).

[0051]

[0052] Among them, the crRNA spacer is 28nt, the 5th base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the wild type or mutant type at the 7th base of the spacer (i.e., 88spacer5-7) can distinguish wild-type and mutant target genes (discrimination ability see Figure 1 ).

[0053]

[0054] Among them, the crRNA spacer is 28nt, the 6th base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the wild type or mutant type at the 8th base of the spacer (i.e., 88spacer6-8) can distinguish wild-type and mutant target genes (discrimination ability see Figure 1 ).

[0055] 2. Preparation of single-site mutation target RNA (ssRNA) and crRNA

[0056] Synthesis of wild-type and mutant target RNAs:

[0057] A plasmid containing a wild-type target gene is synthesized, and site-directed mutagenesis is performed on the plasmid containing the wild-type sequence to obtain a plasmid containing the mutant sequence.

[0058] PCR amplification was performed using primers containing a T7 promoter, and the PCR product was purified.

[0059] The purified gene fragment was used as a template for in vitro RNA transcription using a T7 RNA polymerase Transcription Kit (NEB, E2040s) to obtain ssRNAs with wild-type sequences and ssRNAs containing various mutations.

[0060] Example of a 20 μL transcription system: (1.5 μL each dNTP, 1.5 μL 10× Reaction Buffer, 1.5 μL T7 RNA polymerase Mix, 1 μg template, add enzyme-free water to 20 μL, incubate at 37°C for 16 hours, and finally add 2 μL of DNase I (5 U / μL) (37°C for 15 minutes) to purify the target ssRNA.

[0061] Synthesis of crRNA:

[0062] The single-stranded DNA fragment to be synthesized is determined according to the LwCas13a tracrRNA sequence, direct repeat sequence and designed specific spacer sequence.

[0063] The single-stranded DNA chain and the primer containing T7 RNA polymerase promoter are annealed to form a local double strand, and the product is used as a template for in vitro RNA transcription.

[0064] Example of a 20 μL transcription system: 1.5 μL each dNTP, 1.5 μL 10× Reaction Buffer, 1.5 μL T7 RNA polymerase Mix, 1 μg template, add enzyme-free water to 20 μL, incubate at 37°C for 16 hours, then add 2 μL of DNase I (5 U / μL) (37°C for 15 minutes) to purify crRNA.

[0065] 3. Prokaryotic expression and purification of Cas13a protein

[0066] Using the pC013-Twinstrep-SUMO-huLwCas13a plasmid and Escherichia coli expression system: the pC013-Twinstrep-SUMO-huLwCas13a plasmid vector was transformed into Escherichia coli competent cells by the heat shock method, and positive colonies were picked and inoculated into TB liquid medium for enrichment culture (220 rpm, 37 ° C). When the OD value rose to 0.6, IPTG with a final concentration of 500 μmol / L was added to induce expression, and the culture was continued overnight (220 rpm, 18 ° C).

[0067] E. coli expressing overnight were harvested and resuspended in lysis buffer (20 mmol / L Tris-HCl, pH 8.0, 500 mmol / L NaCl, 1 mmol / L DTT, 1 mg / mL lysozyme, 25 U / mL nuclease and protease inhibitors). After sonication, the supernatant was collected by centrifugation (10,000 g, 1 h). The supernatant was incubated with StrepTactin gel at 4°C for 1 h and centrifuged for 2 min (800 rpm, 4°C). The supernatant was removed and the StrepTactin gel was eluted three times in lysis buffer before being resuspended in digestion buffer (30 mmol / L Tris-HCl, pH 8.0, 500 mmol / L NaCl, 1 mmol / L DTT, 0.15% NP-40). SUMO protease (1 U / μL) was added and incubated overnight at 4°C.

[0068] The supernatant was centrifuged and concentrated, and the supernatant was replaced with buffer (10 mmol / L HEPES, 1 mol / L NaCl, 5 mmol / LMgCl2, 2 mmol / L DTT, pH 7.0) and then subjected to gel filtration chromatography. The purified Cas13a protein was verified by SDS-PAGE electrophoresis and dialyzed overnight at 4 ° C into storage buffer (50 mmol / L Tris-HCl, pH 7.5, 600 mmol / L NaCl, 5% glycerol, 2 mmol / L DTT), and finally frozen in a -80 ° C refrigerator for use.

[0069] 4. Establishment of CRISPR-Cas13a system detection system

[0070] A 50 μL detection reaction system was prepared: 450 nM LwCas13a, 900 nM crRNA, 120 nM fluorescent reporter (Thermo Fisher, RNAse Alert™ QC System v2, or self-prepared RNA-linked fluorescent reporter), 0.5 μL RNase inhibitor (NEB, M0314L), and different amounts of target RNA molecules were added. The entire reaction was carried out in nucleic acid detection buffer (40 mmol / L Tris-HCl, 60 mmol / L NaCl, 6 mmol / L MgCl2, pH 7.3).

[0071] The crRNA, LwCas13a, RNase inhibitor and fluorescent reporter molecule can be prepared into an integrated detection kit.

[0072] The reaction system was incubated at 37°C for 15-60 minutes, during which the fluorescence value was measured every 1 minute. The fluorescence reading was used to determine the RNase activity of Cas13a, indicating the ability of a specific crRNA to guide Cas13a to bind to the target RNA. The wild-type RNA was used as a template and the same crRNA was used to obtain the fluorescence value. The signal ratio obtained by the same crRNA-guided system for recognizing mutant ssRNA and wild-type ssRNA was compared to obtain the ability of a specific crRNA to report a specific single-base mutation.

[0073] 5. Test results

[0074] Results of mutation-specific detection at position 88: Figure 1 The fluorescence value (fluorescence value entering the plateau phase minus the background fluorescence value of the system) corresponding to the mutation of amino acid substitution at position 88 of the GyrA protein was detected using the specified crRNA-guided CRISPR-Cas13a system.

[0075] When the crRNA targeting amino acid 88 of the GyrA protein of Mycobacterium tuberculosis was substituted with the corresponding mutant crRNA to guide the CRISPR-Cas13a system to detect mutant target ssRNA and wild-type ssRNA, the results showed that at 20-30 minutes of detection, the fluorescence signal of the mutant target ssRNA by different crRNA-guided systems was significantly higher than that of the wild-type ssRNA ( Figure 1 In Figures A and B, the signal ratios are 24.45 ± 3.1 for 3-5 crRNA, 38.22 ± 0.91 for 4-6 crRNA, 8.37 ± 0.88 for 5-7 crRNA, and 3.81 ± 0.9 for 6-8 crRNA, respectively. If the ratio of the fluorescence signal obtained by a specific crRNA-guided system recognizing a mutant ssRNA to the signal obtained by its own guide system recognizing a wild-type ssRNA is greater than or equal to 2, the crRNA is considered to be able to distinguish between the mutant and wild-type sequences.

[0076] In summary, the four crRNA-guided CRISPR-Cas13a systems targeting the mutation corresponding to the amino acid substitution at position GyrA88 can distinguish the mutation position, among which crRNA (SEQ ID No. 2) (i.e., 88spacer4-6) has the best detection effect.

[0077] Example 2

[0078] 1. Design of crRNA targeting the 90th amino acid mutation of the gyrA gene of Mycobacterium tuberculosis

[0079] (1) The design principle is the same as that of Example 1.

[0080] (2) Selection of wizard sequence:

[0081] According to the site of mutation, the length of crRNA spacer sequence is 28 nucleotides (nt). C GG T G. Mutation form Ala-Gly base mutation type G C GG G G), 4 crRNA guide sequences targeting it were designed (SEQ ID No. 5 to SEQ ID No. 8). The spacer sequences and their target sequences are shown in the following table, where the bold underlined parts are mutation sites.

[0082]

[0083]

[0084] Among them, the crRNA spacer is 28nt, the third base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the fifth base of the spacer with either the wild type or the mutant type (i.e., 90spacer3-5) can distinguish between wild-type and mutant target genes (discrimination ability see Figure 2 ).

[0085]

[0086] Among them, the crRNA spacer is 28nt, the 4th base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the 6th base of the spacer with the wild type and the mutant type (i.e., 90spacer4-6) can distinguish between wild-type and mutant target genes (discrimination ability see Figure 2 ).

[0087]

[0088] Among them, the crRNA spacer is 28nt, the third base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the fifth base of the spacer with either the wild type or the mutant type (i.e., 90spacer3-5) can distinguish between wild-type and mutant target genes (discrimination ability see Figure 2 ).

[0089]

[0090] Among them, the crRNA spacer is 28nt, the 4th base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the 6th base of the spacer with the wild type and the mutant type (i.e., 90spacer4-6) can distinguish between wild-type and mutant target genes (discrimination ability see Figure 2 ).

[0091] 2. Preparation of single-site mutation target RNA (ssRNA) and crRNA

[0092] The method is the same as Example 1, and the synthesis of mutant target RNA and crRNA is performed for the 90th mutation.

[0093] 3. Prokaryotic expression and purification of Cas13a protein

[0094] Same as Example 1.

[0095] 4. Establishment of CRISPR-Cas13a system detection system

[0096] Same as Example 1.

[0097] 5. Test results

[0098] Results of the 90th mutation-specific test: Figure 2 The fluorescence value (fluorescence value entering the plateau phase minus the background fluorescence value of the system) corresponding to the mutation of amino acid substitution at position 90 of the GyrA protein was detected using the specified crRNA to guide the CRISPR-Cas13a system.

[0099] When the crRNA targeting the 90th amino acid of the GyrA protein of Mycobacterium tuberculosis was substituted with the corresponding mutant, the CRISPR-Cas13a system was used to guide the detection of mutant target ssRNA and wild-type ssRNA. As shown in the results, the fluorescence signal of the mutant target ssRNA by different crRNA-guided systems was significantly higher than that of the wild-type ssRNA at 20-30 minutes of detection ( Figure 2 In Figures A and B, the signal ratios are 8.29±1.51 for A90V 3-5crRNA and 2.94±0.43 for A90V 4-6crRNA; 5.31±1.23 for A90G 3-5crRNA and 7.66±2.35 for A90G 4-6crRNA, respectively. If the ratio of the fluorescence signal obtained by a specific crRNA-guided system recognizing a mutant ssRNA to the signal obtained by its guide system recognizing a wild-type ssRNA is greater than or equal to 2, the crRNA is considered to be able to distinguish between the mutant and wild-type sequences.

[0100] In summary, the four crRNAs targeting the mutation corresponding to the GyrA90 amino acid substitution guided the CRISPR-Cas13a system to distinguish the mutation position, among which the A90V type mutation crRNA (SEQ ID No. 5) (i.e., A90V spacer 3-5) had the best detection effect, and the A90G type mutation crRNA (SEQ ID No. 8) (i.e., A90G spacer 4-6) had the best detection effect.

[0101] Example 3

[0102] 1. Design of crRNA targeting the 91st amino acid mutation of the gyrA gene of Mycobacterium tuberculosis

[0103] (1) The design principle is the same as that of Example 1.

[0104] (2) Selection of wizard sequence:

[0105] According to the site of mutation, the length of crRNA spacer sequence is 28 nucleotides (nt). T CG- C CG), a crRNA guide sequence targeting it was designed (SEQ ID No. 9). The spacer sequence and the target sequence it targets are shown in the following table, where the bold underlined part is the mutation site.

[0106]

[0107] Among them, the crRNA spacer is 28 nt, the 10th base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the wild type or mutant type at the 17th base of the spacer (i.e., 91spacer10-17) can distinguish wild-type and mutant target genes (discrimination ability see Figure 3 ).

[0108] 2. Preparation of single-site mutation target RNA (ssRNA) and crRNA

[0109] The method was the same as in Example 1, except that the synthesis of the mutant target RNA and crRNA was performed for the 91st mutation.

[0110] 3. Prokaryotic expression and purification of Cas13a protein

[0111] Same as Example 1.

[0112] 4. Establishment of CRISPR-Cas13a system detection system

[0113] Same as Example 1.

[0114] 5. Test results

[0115] Results of mutation-specific detection at position 91: Figure 3 The fluorescence value (fluorescence value entering the plateau phase minus the background fluorescence value of the system) corresponding to the mutation of amino acid substitution at position 91 of the GyrA protein was detected using the specified crRNA to guide the CRISPR-Cas13a system.

[0116] When the crRNA was designed by replacing the corresponding mutation at amino acid position 91 of the GyrA protein of Mycobacterium tuberculosis to guide the CRISPR-Cas13a system to detect mutant target ssRNA and wild-type ssRNA, as shown in the results, the fluorescence signal of the crRNA-guided system for the mutant target ssRNA was significantly higher than that for the wild-type ssRNA at 20-30 minutes of detection ( Figure 3 In Figures A and B, the signal ratios are 10-17 crRNA:11.98±0.21, respectively. If the ratio of the fluorescence signal obtained by a specific crRNA-guided system recognizing a mutant ssRNA to the signal obtained by its guide system recognizing a wild-type ssRNA is greater than or equal to 2, the crRNA is considered to be able to distinguish between the mutant sequence and the wild-type sequence.

[0117] In summary, the crRNA-guided CRISPR-Cas13a system targeting the mutation corresponding to the amino acid substitution at position 91 of GyrA can distinguish the mutation position, and the detection effect of crRNA (SEQ ID No. 9) (i.e., 91 spacer 10-17) is good.

[0118] Example 4

[0119] 1. Design of crRNA targeting the 94th amino acid mutation of the gyrA gene of Mycobacterium tuberculosis

[0120] (1) The design principle is the same as that of Example 1.

[0121] (2) Selection of wizard sequence:

[0122] According to the site of mutation, the length of crRNA spacer sequence is 28 nucleotides (nt). G AC- A AC, mutant form Asp-His base mutation type G AC- C AC, mutation form Asp-Tyr base mutation typeG AC- T AC and mutant form Asp-Gly base mutation type G A CG G C), four crRNA guide sequences targeting it were designed (SEQ ID No. 10 to SEQ ID No. 13). The spacer sequences and their target sequences are shown in the following table, where the bold underlined parts are mutation sites.

[0123]

[0124] Among them, the crRNA spacer is 28 nt, the 10th base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the wild type or mutant type at the 16th base of the spacer (i.e., 94spacer10-16) can distinguish wild-type and mutant target genes (discrimination ability see Figure 4 ).

[0125]

[0126]

[0127] Among them, the crRNA spacer is 28 nt, the 10th base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the wild type or mutant type at the 16th base of the spacer (i.e., 94spacer10-16) can distinguish wild-type and mutant target genes (discrimination ability see Figure 4 ).

[0128]

[0129] Among them, the crRNA spacer is 28 nt, the 10th base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the wild type or mutant type at the 16th base of the spacer (i.e., 94spacer10-16) can distinguish wild-type and mutant target genes (discrimination ability see Figure 4 ).

[0130]

[0131] Among them, the crRNA spacer is 28 nt, the 17th base of the spacer is complementary to the mutant type and does not match the wild type, and the type of crRNA that does not match the wild type or mutant type at the 10th base of the spacer (i.e., 94spacer17-10) can distinguish between wild-type and mutant target genes (discrimination ability see Figure 4 ).

[0132] 2. Preparation of single-site mutation target RNA (ssRNA) and crRNA

[0133] The method is the same as Example 1, and the synthesis of mutant target RNA and crRNA is performed for the 94th mutation.

[0134] 3. Prokaryotic expression and purification of Cas13a protein

[0135] Same as Example 1.

[0136] 4. Establishment of CRISPR-Cas13a system detection system

[0137] Same as Example 1.

[0138] 5. Test results

[0139] Results of mutation-specific detection at position 94: Figure 4 The fluorescence value (fluorescence value entering the plateau phase minus the background fluorescence value of the system) corresponding to the mutation of amino acid substitution at position 94 of the GyrA protein was detected using the specified crRNA-guided CRISPR-Cas13a system.

[0140] When the crRNA was designed by replacing the corresponding mutation at amino acid position 94 of the GyrA protein of Mycobacterium tuberculosis to guide the CRISPR-Cas13a system to detect mutant target ssRNA and wild-type ssRNA, as shown in the results, the fluorescence signal of the crRNA-guided system for the mutant target ssRNA was significantly higher than that for the wild-type ssRNA at 20-30 minutes of detection ( Figure 4 In Figures A and B, the signal ratios are 15.09±0.64 for D94N 10-16 crRNA; 36.9±1.63 for D94H 10-16 crRNA; 35.77±4.78 for D94T 10-16 crRNA; and 9.29±0.73 for D94G 17-10 crRNA. A ratio of the fluorescence signal obtained by a specific crRNA-guided system recognizing a mutant ssRNA to the signal obtained by its guide system recognizing a wild-type ssRNA is greater than or equal to 2, indicating that the crRNA can distinguish between the mutant and wild-type sequences.

[0141] In summary, the CRISPR-Cas13a system targeting the amino acid substitution at position 94 of GyrA corresponding to the mutant crRNA can distinguish the mutation position, the detection effect of mutant type D94N crRNA (SEQ ID No. 10) (i.e. D94N spacer 10-16) is the best, the detection effect of mutant type D94H crRNA (SEQ ID No. 11) (i.e. D94H spacer 10-16) is the best, the detection effect of mutant type D94T crRNA (SEQ ID No. 12) (i.e. D94T spacer 10-16) is the best, and the detection effect of mutant type D94G crRNA (SEQ ID No. 13) (i.e. D94G spacer 17-10) is the best.

[0142] Example 5

[0143] 1. Design of crRNA targeting the mutation of amino acid at position 88 of gyrA gene of Mycobacterium tuberculosis (negative control experiment)

[0144] (1) The design principle is the same as that of Example 1.

[0145] (2) Selection of guide sequence:

[0146] According to the mutation site, the length of the crRNA spacer is 28 nucleotides (nt). For the mutation at position 88 (mutation form Gly-Ala base mutation type G G C-G C C), a crRNA guide sequence (SEQ ID No. 36) targeting it is designed, and the spacer and its target sequence are shown in the following table, wherein the bold underlined part is the mutation site.

[0147]

[0148] Among them, the spacer of crRNA is 28 nt, the bases of spacer are completely complementary to the mutant type, and there is no base mismatch crRNA type (reaction kinetics curve see Figure 5 ).

[0149] 2. Preparation of single-site mutant RNA (ssRNA) and crRNA

[0150] The method is the same as that of Example 1. The synthesis of mutant target RNA and the synthesis of crRNA are carried out for the mutation at position 88.

[0151] 3. Prokaryotic expression and purification of Cas13a protein

[0152] The same as Example 1.

[0153] 4. Establishment of CRISPR-Cas13a system detection system

[0154] Same as Example 1.

[0155] 5. Test results

[0156] Results of mutation detection at position 88: Figure 5 Fluorescence kinetic curves of the mutation corresponding to the amino acid substitution at position 88 of the GyrA protein using the specified crRNA-guided CRISPR-Cas13a system and the negative control reaction curves of the reactions (experiments without adding crRNA, Cas13a or ssRNA, respectively).

[0157] When the crRNA targeting amino acid 88 of the GyrA protein of Mycobacterium tuberculosis was substituted with the corresponding mutant crRNA to guide the CRISPR-Cas13a system to detect the mutant target ssRNA, as shown in the results, the fluorescence signal value of the crRNA-guided system for the mutant target ssRNA reached a plateau at 30-40 minutes of detection ( Figure 5 In the assay, the fluorescence signal values ​​were Cas13a / crRNA+Target RNA: 9404.72±968.35; Cas13a / crRNA+nontarget RNA: 552.33±6.33; Cas13a+Target RNA: 544.44±3.08; crRNA+Target RNA: 547.44±11.77).

[0158] Detection sensitivity results

[0159] The integrated detection system provides a sensitivity of 1×100 copies / μL to 1×10 2 copies / μL.

[0160] Clinical performance evaluation

[0161] A total of 75 clinical strains of Mycobacterium tuberculosis were collected, and 35 fluoroquinolone-resistant strains (resistant to ofloxacin or levofloxacin) and 40 quinolone-sensitive strains were identified by culture-based methods. The genomic DNA of the strains was isolated and added to the detection system in an amount of 2 ng. Mutation detection was performed using crRNA targeting different mutations according to the method described above. The method established in this article detected 32 fluoroquinolone-associated mutations, and the remaining 43 Mycobacterium tuberculosis genomic DNAs were determined by the system to contain no fluoroquinolone-associated mutations. The results are shown in the table below. The genomic DNA was expanded using PCR in the QRDR region and sequenced. The resistance information obtained by the CRISPR-Cas13a method was compared with the DNA sequencing results. The former had a 100% consistency with the latter. Based on the culture sensitivity results, it was judged that the three inconsistent strains were quinolone-tolerant but sensitive by the CRISPR-Cas13a method. DNA sequencing found that there were no mutations in the corresponding positions. The three strains were phenotypically resistant strains.

[0162] Table 1. Summary of clinical evaluation results

[0163]

[0164] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. SEQUENCE LISTING <110> Chongqing Medical University International In Vitro Diagnostics Research Institute <120> crRNA, kit, and detection method for detecting mutations in the gyrA fluoroquinolone-resistance-related gene of Mycobacterium tuberculosis <130> PA20024796 <160> 36 <170> PatentIn version 3.3 <210> 1 <211> 28 <212> RNA <213> crRNA spacer <400> 1 cggccugcgg gugguaguug cccauggu 28 <210> 2 <211> 28 <212> RNA <213> crRNA spacer <400> 2 ucggccugcg ggugguaguu gcccaugg 28 <210> 3 <211> 28 <212> RNA <213> crRNA spacer <400> 3 gucggccugc gggugguagu ugcccaug 28 <210> 4 <211> 28 <212> RNA <213> crRNA spacer <400> 4 cgucggccug cgggugguag uugcccau 28 <210> 5 <211> 28 <212> RNA <213> crRNA spacer <400> 5 acaccucgcc gugcgggugg uaguugcc 28 <210> 6 <211> 28 <212> RNA <213> crRNA spacer <400> 6 gacaccucgc cgugcgggug guaguugc 28 <210> 7 <211> 28 <212> RNA <213> crRNA spacer <400> 7 accccucgcc gugcgggugg uaguugcc 28 <210> 8 <211> 28 <212> RNA <213> crRNA spacer <400> 8 gaccccucgc cgugcgggug guaguugc 28 <210> 9 <211> 28 <212> RNA <213> crRNA spacer <400> 9 cguagaucgg cgcguccccg ugcgggug 28 <210> 10 <211> 28 <212> RNA <213> crRNA spacer <400> 10 ccaggcuguu guagaacgac gcgucgcc 28 <210> 11 <211> 28 <212> RNA <213> crRNA spacer <400> 11 ccaggcugug guagaacgac gcgucgcc 28 <210> 12 <211> 28 <212> RNA <213> crRNA spacer <400> 12 ccaggcugua guagaacgac gcgucgcc 28 <210> 13 <211> 28 <212> RNA <213> crRNA spacer <400> 13 caugcgcacg aggcugccgu agaucgac 28 <210> 14 <211> 28 <212> RNA <213> 野生型ssRNA <400> 14 accaugggca acuaccaccc gcacggcg 28 <210> 15 <211> 28 <212> RNA <213> mutant ssRNA <400> 15 accaugggca acuaccaccc gcacgccg 28 <210> 16 <211> 28 <212> RNA <213> Wild-type ssRNA <400> 16 ccaugggcaa cuaccacccg cacggcga 28 <210> 17 <211> 28 <212> RNA <213> mutant ssRNA <400> 17 ccaugggcaa cuaccacccg cacgccga 28 <210> 18 <211> 28 <212> RNA <213> Wild-type ssRNA <400> 18 caugggcaac uaccacccgc acggcgac 28 <210> 19 <211> 28 <212> RNA <213> mutant ssRNA <400> 19 caugggcaac uaccacccgc acgccgac 28 <210> 20 <211> 28 <212> RNA <213> Wild-type ssRNA <400> 20 augggcaacu accacccgca cggcgacg 28 <210> twenty one <211> 28 <212> RNA <213> mutant ssRNA <400> twenty one augggcaacu accacccgca cgccgacg 28 <210> twenty two <211> 28 <212> RNA <213> Wild-type ssRNA <400> twenty two ggcaacuacc acccgcacgg cgacgcgu 28 <210> twenty three <211> 28 <212> RNA <213> mutant ssRNA <400> twenty three ggcaacuacc acccgcacgg cgacgugu 28 <210> twenty four <211> 28 <212> RNA <213> Wild-type ssRNA <400> twenty four gcaacuacca cccgcacggc gacgcguc 28 <210> 25 <211> 28 <212> RNA <213> mutant ssRNA <400> 25 gcaacuacca cccgcacggc gacguguc 28 <210> 26 <211> 28 <212> RNA <213> mutant ssRNA <400> 26 ggcaacuacc acccgcacgg cgacgggu 28 <210> 27 <211> 28 <212> RNA <213> mutant ssRNA <400> 27 gcaacuacca cccgcacggc gacgggguc 28 <210> 28 <211> 28 <212> RNA <213> Wild-type ssRNA <400> 28 cacccgcacg gcgacgcguc gaucuacg 28 <210> 29 <211> 28 <212> RNA <213> mutant ssRNA <400> 29 cacccgcacg gcgacgcgcc gaucuacg 28 <210> 30 <211> 28 <212> RNA <213> Wild-type ssRNA <400> 30 ggcgacgcgu cgaucuacga cagccugg 28 <210> 31 <211> 28 <212> RNA <213> mutant ssRNA <400> 31 ggcgacgcgu cgaucuacaa cagccugg 28 <210> 32 <211> 28 <212> RNA <213> mutant ssRNA <400> 32 ggcgacgcgu cgaucuacca cagccugg 28 <210> 33 <211> 28 <212> RNA <213> mutant ssRNA <400> 33 ggcgacgcgu cgaucuacua cagccugg 28 <210> 34 <211> 28 <212> RNA <213> Wild-type ssRNA <400> 34 gucgaucuac gacagccugg ugcgcaug 28 <210> 35 <211> 28 <212> RNA <213> mutant ssRNA <400> 35 gucgaucuac ggcagccugg ugcgcaug 28 <210> 36 <211> 28 <212> RNA <213> crRNA spacer <400> 36 cggcgugcgg gugguaguug cccauggu 28

Claims

1. Detecting crRNA for fluoroquinolone resistance gene mutations in Mycobacterium tuberculosis, characterized in that The spacer sequence of the crRNA is any one of SEQ ID No.1 to SEQ ID No.

13.

2. A CRISPR-Cas13a system, characterized in that Comprising the crRNA as described in claim 1.

3. The CRISPR-Cas13a system according to claim 2, wherein Also included are a plasmid capable of expressing Cas13a protein, a signal reporter probe, and a buffer.

4. The CRISPR-Cas13a system according to claim 3, wherein The Cas13a protein is selected from LwCas13a or LshCas13a.

5. The CRISPR-Cas13a system according to claim 4, wherein The Cas13a protein is LwCas13a.

6. The crRNA according to claim 1 or the CRISPR-Cas13a system according to any one of claims 2 to 5 for non-disease diagnosis purposes of Mycobacterium tuberculosis gyrA Application in gene mutation detection.

7. A kit, characterized in that The kit contains the crRNA as described in claim 1; the kit is used to detect fluoroquinolone-resistant gene mutations in Mycobacterium tuberculosis.

8. Mycobacterium tuberculosis for non-disease diagnosis purposes gyrA A method for detecting gene mutation, characterized in that: The method comprises: obtaining a sample RNA to be tested; incubating the crRNA according to claim 1, the sample RNA, the LwCas13a protein, a fluorescently labeled probe, and an RNase inhibitor in a buffer system and then detecting.

9. The method according to claim 8, characterized in that After the buffer system is incubated, it is added to the sample detection area of ​​the colloidal gold test strip for detection, or the fluorescence value is measured by a fluorescence detector after the system is incubated.

10. The method according to claim 9, characterized in that Fluorescence was detected every 1–2 min on a microplate reader.

11. The method according to claim 8, characterized in that The contents of each component in the buffer system are: LwCas13a protein 400-500 nM, crRNA 800-1000 nM, sample RNA 1600-2000 nM, fluorescently labeled probe 120-200 nM and RNase inhibitor 0.5 μL.

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