Multiple CRISPR nucleic acid detection technology based on crRNA metrological coding

Abstract

The invention discloses a multiple CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) nucleic acid detection technology based on crRNA metrology coding, and belongs to the technical field of biology. According to the technical method disclosed by the invention, specific crRNA of CRISPR is designed aiming at different nucleic acid targets, coding is carried out through crRNA with different concentrations, and the fluorescence intensity is obtained based on a CRISPR multiple detection technology. The strength of the CRISPR signal is determined by the number of Cas12a / crRNA activated by the target, so that single-tube detection of multiple targets can be realized through the fluorescence intensity coded by the crRNA. According to the detection method, nucleic acid CRISPR multiple detection is achieved, specific types of genotypes are achieved through a logic gate, and the method has the advantages of being simple, convenient and rapid in multiple detection and can be used for detecting different types of nucleic acid targets related to disease diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a multiple CRISPR nucleic acid detection technology based on crRNA metrological coding. Background Art

[0002] Emerging human, animal and plant diseases are one of the major threats to global health and human civilization. Early, rapid and effective disease detection is essential to maximize crisis management efficiency, treatment outcomes and economic stability. However, the current practice of human, animal and plant disease detection is mainly limited to centralized laboratories. Typically, patients or samples are taken to professional institutions such as hospitals or testing companies for testing, and the test results take several days to return. Fast and convenient nucleic acid testing technology is essential to improve epidemic prevention and control. In addition, in the field of daily medical activities, nucleic acid testing technology plays an indispensable and important role. In terms of infectious diseases, nucleic acid testing can quickly determine the type of pathogens and start treatment early, thereby reducing the mortality and complications associated with infectious diseases. In the field of chronic diseases and cancer, prognosis-related genetic testing is often inseparable from prognosis and determines the choice of subsequent treatment options. Nucleic acid testing also plays an important role in animal quarantine and plant disease determination. The development of fast, convenient and low-cost nucleic acid testing technology is urgently needed.

[0003] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated CRISPR-associated protein (Cas) system have efficient nucleic acid targeting recognition capabilities and have become a new generation of molecular diagnostic technology, especially showing superior performance in the field of nucleic acid detection. However, CRISPR-based nucleic acid detection technology has shortcomings in multiple detection, such as long detection time and cumbersome detection steps, and generally cannot achieve single-tube, single-label detection of multiple targets. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a multiple CRISPR nucleic acid detection technology based on crRNA metrological coding. For the first time, CRISPR multiple detection of nucleic acids is achieved by metrological coding of crRNA, and genotyping can be achieved. We use different concentrations of crRNA for encoding different nucleic acid targets. The strength of the CRISPR signal is determined by the number of activated Cas12a / crRNA, so CRISPR multiple detection can be achieved by the fluorescence intensity encoded by crRNA.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The present invention discloses a multiple CRISPR nucleic acid detection method based on crRNA quantitative coding, characterized in that the method targets the nucleic acid sequence of a target gene, designs specific crRNA primers based on CRISPR, and performs encoding through crRNA of different concentrations to achieve multiple gene detection for different typing of the target gene.

[0007] Furthermore, the method also includes realizing multiple genotyping detection with logical relationships through logic gate operations.

[0008] Furthermore, the specific operation steps of the method include: Step 1: Based on the CRISPR method, specific crRNA primers are designed for the target gene and its genotyping; Step 2, establishing a multiplex gene detection reaction system and conditions for detection; Step 3: Calculate the fluorescence endpoint value based on the fluorescence amplification curve, and perform qualitative and quantitative detection of sample genotyping based on the fluorescence endpoint value.

[0009] Furthermore, the multiple gene detection reaction system in step 2 includes: a test sample, Cas12a, a reporter, NEBuffer r2.1, a crRNA primer and ddH2O.

[0010] Furthermore, the reaction conditions of the multiple gene detection in step 2 are: 37° C., reaction time 30 to 45 minutes.

[0011] On the other hand, the present invention discloses a multiple CRISPR nucleic acid detection system based on crRNA quantitative coding, characterized in that the nucleic acid detection system comprises specific crRNA primers for the nucleic acid sample to be tested, Cas12a, a buffer and a reporter.

[0012] Furthermore, the system also includes realizing multiple genotyping detection with logical relationships through logic gate operations.

[0013] The third aspect of the present invention discloses a multiple CRISPR nucleic acid detection kit based on crRNA metrological coding, characterized in that the kit comprises the multiple CRISPR nucleic acid detection system based on crRNA metrological coding as described in any one of the above items.

[0014] The fourth aspect of the present invention discloses a multiple CRISPR nucleic acid detection method based on crRNA metrological coding as described above, or a multiple CRISPR nucleic acid detection system based on crRNA metrological coding as described above, or a multiple CRISPR nucleic acid detection kit based on crRNA metrological coding as described above, applied to genotyping detection.

[0015] Furthermore, the application can realize single-tube, single-Cas enzyme, single-marker CRISPR multiple genotyping detection through the quantitative coding of crRNA.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] The present invention uses linear concentration and logic gate operations to perform metering and coding of crRNA concentration to achieve signal intensity differentiation of different targets, thereby being used for nucleic acid multiplex detection and for genotyping. Single-tube, single-Cas enzyme, single-label CRISPR multiplex detection can be achieved by metering and coding of crRNA.

[0018] The present invention can not only realize general nucleic acid multiplex detection genotyping through general linear crRNA encoding, but also realize nucleic acid multiplex detection genotyping with logical relationship through logic gate crRNA encoding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Brucella spp. using crRNA-encoded CRISPR multiplex detection omp2a Schematic diagram of the genotyping principle.

[0020] Figure 2 Three Brucella species for multiplex CRISPR reactions B. abortus , melitensis , B.suis Genotyping. A is the fluorescence curve graph; B is the bar graph obtained based on the end point fluorescence value of Figure A.

[0021] Figure 3 Brucella omp2a Multiple CRISPR detection of real genetic samples.

[0022] Figure 4 Schematic diagram of the principle of ABO genotyping using crRNA-encoded CRISPR multiplex detection.

[0023] Figure 5 ABO genotyping for crRNA-encoded CRISPR "AND" logic gate multiplex detection. A is a fluorescence curve graph; B is a bar graph obtained based on the end point fluorescence value of Figure A.

[0024] Figure 6 Perform blood typing tests on real samples for multiplex CRISPR systems. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0026] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0027] Different crRNA encodings are performed for different target genes, and the gene typing can be effectively performed through the strength of the output signal.

[0028] The reaction system of the method described in the present invention includes: target DNA, multiple crRNA sequences, Cas12a protein, fluorescent probes modified by fluorescent groups and quenching groups, and reaction buffer.

[0029] The specific operation process of the method of the present invention.

[0030] Step 1: Based on the CRISPR method, specific crRNA primers are designed for the target gene and its genotyping; Step 2, establishing a multiplex gene detection reaction system and conditions for detection; The reaction system includes: a test sample, Cas12a, a reporter, NEBuffer r2.1, a crRNA primer and ddH2O; The reaction conditions are: 37°C for 30-45 min; Step 3: Calculate the fluorescence endpoint value based on the fluorescence amplification curve, and perform qualitative and quantitative detection of sample genotyping based on the fluorescence endpoint value. Example 1 The CRISPR multiplex detection system based on crRNA quantitative coding described in the present invention is used to type Brucella genes.

[0031] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0032] This embodiment includes the following steps.

[0033] (1) Brucella omp2a The plasmids of the genes were amplified by PCR. omp2a The three subtype amplification products of the gene are B.ab, B.me and B.su.

[0034] (2) Specific crRNA primers were designed for the three subtypes of Brucella genes: abcrRNA, mecrRNA and sucrRNA (sequence information is shown in Table 1).

[0035] Table 1. crRNA sequences encoding three subtypes of Bushella .

[0036] (3) Establish a CRISPR multi-target detection system to type Brucella genes. Cas12a (1μM) 0.5μL, reporter (1μM) 2μL, 2μL NEBuffer r2.1 (10×), 1μL of the PCR product amplified from the plasmid in step (1) as the detection target, add different concentrations of crRNA (1nM abcrRNA, 25nM mecrRNA, 100nM sucrRNA) from step (2), make up to 20μL with water, and then place it in a real-time fluorescence quantitative PCR instrument for detection. The reaction temperature is 37°C and the reaction time is 45min. Figure 2 Figure A shows the fluorescence amplification curves of three Brucella genes; Figure (B) corresponds to the fluorescence endpoint value of Figure (A). The final fluorescence value is above 18.51. B. suis The final fluorescence value is between 12.21-18.51. B. melitensis The final fluorescence value is between 4.07-12.21. B. abortus , if the value is below 4.07, there is no such gene target.

[0037] (4) omp2a Gene detection in real samples. omp2a Genetic E. coli The samples were spiked with human serum to simulate Brucella "infected serum" and tested for 25 samples. The reaction system was configured according to step (3) and the endpoint value of the fluorescence curve of the actual sample was measured by real-time fluorescence quantitative PCR instrument ( Figure 3 ). 1-5 is B. abortus , 6-10 B. melitensis : 6-10, 11-15 B.suis , 16-20 E. coli , 21-25 are ctrlserum). They all conform to the range of the three subtypes divided in step (3).

[0038] Example 2 ABO blood type genotyping is performed based on multiple detection of CRISPR logic gates encoded by crRNA.

[0039] This example introduces logic gates into the crRNA encoding strategy and successfully performs ABO blood typing by establishing a CRISPR "AND" logic gate system encoded by crRNA. Figure 4As shown, the A, B, and AB sequences in the ABO gene are amplified by ARMS-PCR. Since the O sequence does not match the 3′ of the primer, amplification cannot be performed. When the system contains a combined target of AB blood type, the two Cas12a / AcrRNA and Cas12a / BcrRNA complexes in the system are activated at the same time, and the fluorescence intensity is the sum of the activated targets, so that the AB blood type is distinguished by the additive relationship, while the fluorescence intensity of the A and B blood types is determined by the concentration of their respective crRNA encodings. From the schematic diagram ( Figure 4 ) It can be seen that the CRISPR reaction produces a low fluorescence signal for the A blood type sequence, a medium fluorescence signal for the B blood type sequence, and a high fluorescence signal for the sum of the two sequences activated in the system, which is the AB sequence.

[0040] 1. Steps.

[0041] (1) Perform PCR amplification on the plasmid containing the ABO blood type gene (PCR primer sequences are shown in Table 2), obtain the A gene amplification product as the A gene target (A), obtain the B gene amplification product as the B gene target (B), obtain the AB gene amplification product as the target for detecting the AB gene (AB), and do not amplify the O gene.

[0042] (2) Design specific crRNA primers for gene A and gene B: AbcrRNA and BcrRNA (see Table 2).

[0043] Table 2 crRNA sequences and PCR primer sequences for CRISPR detection of ABO gene .

[0044] (3) crRNA-encoded CRISPR “AND” logic gate for multiplex detection of ABO blood type genes. Set the “AND” logic gate AB fluorescence signal increment = A fluorescence signal increment + B fluorescence signal increment.

[0045] Multiplex CRISPR reaction system: NEBuffer r2.1 (10×) 2μL, AcrRNA (0.5nM) 1μL, BcrRNA (60nM) 1μL, Cas12a (1μM) 1.5μL, Reporter (1μM) 4μL, target (plasmid PCR amplification product in step (1)) 1μL, and water was used to make up to 20μL. The reaction temperature was 37°C, and the fluorescence endpoint value of the reaction for 30 minutes was above 0.2040 for AB gene, between 0.2040-0.1824 for B gene, between 0.1384-0.1824 for A gene, and below 0.1384 for O gene. Subsequent experiments used this boundary statistical data for ABO genotyping.

[0046] (4) The crRNA-encoded CRISPR “AND” logic gate multiplex detection method was used to perform blood typing tests on actual samples. In this study, oral epithelial cells of 8 different blood types were extracted, PCR amplification was performed according to step (1), and a multiplex CRISPR reaction system was configured according to step (3). The real-time fluorescence quantitative PCR instrument was used for detection and signal reading, and the fluorescence time curve ( Figure 6 ) to verify the typing results. Samples 1-3 and 13-15 with a final fluorescence value of more than 0.2040 are AB blood type; samples 4-6 and 16-18 with a final fluorescence value of 0.1824-0.2040 are B blood type; samples 7-9 and 19-21 with a final fluorescence value of 0.1384-0.1824 are A blood type. The remaining samples 10-12 and 22-24 are below 0.1433 and are O blood type. The final interpretation results are consistent with the actual samples.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the content and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A multiplex CRISPR nucleic acid detection method based on crRNA quantitative coding, characterized in that: The method designs specific crRNA primers based on CRISPR for the target gene nucleic acid sequence, and performs encoding through crRNA of different concentrations to achieve multiple gene detection for different typing of the target gene.

2. The detection method according to claim 1, characterized in that: The method also includes realizing multiple genotyping detection with logical relationships through logic gate operations.

3. The multiple CRISPR nucleic acid detection method based on crRNA quantitative coding according to claim 1, characterized in that: The specific operation steps of the method include: Step 1: Based on the CRISPR method, specific crRNA primers are designed for the target gene and its genotyping; Step 2, establishing a multiplex gene detection reaction system and conditions for detection; Step 3: Calculate the fluorescence endpoint value based on the fluorescence amplification curve, and perform qualitative and quantitative detection of sample genotyping based on the fluorescence endpoint value.

4. The step according to claim 3, characterized in that: The multiple gene detection reaction system in step 2 includes: a detection sample, Cas12a, a reporter, NEBuffer r2.1, a crRNA primer and ddH2O.

5. The step according to claim 3, characterized in that: The reaction conditions of the multiple gene detection in step 2 are: 37° C., reaction time 30-45 min.

6. A multiplex CRISPR nucleic acid detection system based on crRNA quantitative coding, characterized in that: The nucleic acid detection system includes specific crRNA primers for the nucleic acid sample to be tested, Cas12a, a buffer and a reporter.

7. The detection system according to claim 6, characterized in that: The system also includes realizing multiple genotyping detection with logical relationship through logic gate operation.

8. A multiplex CRISPR nucleic acid detection kit based on crRNA quantitative coding, characterized in that: The kit comprises a multiple CRISPR nucleic acid detection system based on crRNA metrological encoding as described in any one of claims 6 to 7.

9. A multiple CRISPR nucleic acid detection method based on crRNA metrological encoding according to any one of claims 1 to 5, or a multiple CRISPR nucleic acid detection system based on crRNA metrological encoding according to any one of claims 6 to 7, or a multiple CRISPR nucleic acid detection kit based on crRNA metrological encoding according to claim 8, applied to genotyping detection.

10. The use according to claim 9, characterized in that: The application can realize single-tube, single-Cas enzyme, single-marker CRISPR multiple genotyping detection through the quantitative coding of crRNA.

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