WO2022257659A1 - Procédé et kit de détection rapide de sars-cov-2 à base de système crispr/cas - Google Patents

Procédé et kit de détection rapide de sars-cov-2 à base de système crispr/cas Download PDF

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WO2022257659A1
WO2022257659A1 PCT/CN2022/091168 CN2022091168W WO2022257659A1 WO 2022257659 A1 WO2022257659 A1 WO 2022257659A1 CN 2022091168 W CN2022091168 W CN 2022091168W WO 2022257659 A1 WO2022257659 A1 WO 2022257659A1
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crispr
solution
nucleic acid
concentration
crrna
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杜忆南
邢晨
柳燕
周静
祝亚亭
王小凤
李倩
张智康
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安徽医科大学
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the invention belongs to the technical field of biological detection, and relates to a novel coronavirus dual-target rapid detection method and a kit based on a CRISPR/Cas system.
  • Novel coronavirus (COVID-19), referred to as "new coronavirus”, is a newly discovered single-stranded RNA virus with a total length of 29,903 nucleotides. It is transmitted through the respiratory tract and conjunctiva through droplets. It is highly infectious and spreads widely. , is currently the seventh coronavirus known to be pathogenic to humans. Compared with the acute symptoms caused by other coronaviruses, the symptoms of 2019-nCoV infection range from mild, cough, fever to critical illness. The symptoms of infection are similar to common respiratory diseases, and they are highly concealed and highly contagious.
  • the new crown vaccine requires a long period of time. After the development of the vaccine, the universal vaccination and the formation of immunity also require a huge vaccine production capacity and a very long vaccination cycle. Therefore, the most effective way to prevent and control the new coronavirus is still early detection and early isolation, through nucleic acid testing to find people infected with the new coronavirus, and isolate the infected people and related close contacts to block the spread of the virus.
  • the gold standard for nucleic acid diagnosis of the new coronavirus is real-time fluorescent quantitative PCR (qPCR).
  • This method amplifies the target fragment of the new coronavirus nucleic acid by PCR, and cuts and breaks a taq-man probe every time a target fragment is generated during amplification to generate a fluorescent signal .
  • the generation of fluorescent signal is synchronized with the amplification product. Since the template increases exponentially during PCR amplification, there is a logarithmic relationship between the number of cycles (Ct) performed when it reaches the set threshold and the reciprocal of the initial template amount, and quantitative analysis can be performed based on this, thereby realizing the quantitative detection of the new coronavirus.
  • Ct number of cycles
  • qPCR requires expensive thermal cyclers and skilled operators, and the detection cycle is generally 3-4 hours long, which is difficult to meet the screening needs in some special scenarios, such as epidemic prevention and control in resource-poor areas such as remote rural areas.
  • RT-LAMP reverse transcription-loop-mediated isothermal amplification
  • RT-RPA-exo probe method reverse transcription-recombinase polymerase amplification exo probe method
  • CRISPR is the abbreviation of "Clustered regularly interspaced short palindromic repeats”, which refers to regularly clustered interspaced short palindromic repeats.
  • Cas is the abbreviation of "CRISPR-associated”, which is related to CRISPR.
  • the CRISPR/Cas system is an adaptive mechanism evolved by bacteria and archaea to resist the invasion of phages. It was discovered and developed into a technology in which guide RNA guides Cas nucleases to perform specific nucleic acid editing on targeted genes.
  • crRNA CRISPR-derived RNA
  • tracrRNA trans-activating RNA
  • tracrRNA trans-activating RNA
  • the double-stranded DNA is cut at the target site, so as to realize the editing of the genomic DNA sequence; and by artificially designing these two RNAs, it can be transformed into a guide RNA (guide RNA), which is enough to guide Cas9 to cut DNA at a specific point.
  • guide RNA guide RNA
  • the rapid nucleic acid detection technology based on CRISPR is mainly divided into two categories, that is, the nucleic acid constant temperature detection technology called DETECTR developed by Jennifer Doudna, the winner of the 2020 Nobel Prize in Chemistry, which relies on Cas12a, and the technology developed by Zhang Feng, the owner of the CRISPR patent.
  • a nucleic acid isothermal detection technology called SHERLOCK that relies on Cas13a. The principle is to amplify and enrich the target fragment through a constant temperature amplification method such as RPA.
  • the amplified target fragment will be targeted and recognized by the Cas protein through a piece of crRNA guidance, and the Cas protein will be activated to become a DNA cutting machine (Cas12a).
  • RNA cutter which cuts all nearby single-stranded DNA (Cas12a) or single-stranded RNA (cas13a). This feature acts on single-stranded nucleic acid fluorescent probes and can be used to report detection results.
  • the DETECTR system and the SHERLOCK system can only detect a single site at a time, and cannot detect double or even triple targets by adding taq-man probes of different target sites like qPCR. When encountering some low-copy clinical samples, the multiple-target detection method can avoid false negatives caused by the incomplete nucleic acid genome of the new coronavirus.
  • the DETECTR and SHERLOCK systems based on the CRISPR/Cas system can only detect a single target at present.
  • each different Cas protein can only cut the corresponding single-stranded DNA probe (LbaCas12a) or single-stranded RNA probe (LwaCas13a) after being activated, which makes the integration of the two CRISPR systems into a single system for dual-target detection. Simultaneous detection is possible.
  • the object of the present invention is to provide a novel coronavirus dual-target rapid detection method and kit based on the CRISPR/Cas system.
  • a CRISPR/Cas system-based kit for rapid double-target detection of novel coronavirus comprising:
  • Cas13-S-for 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';
  • Cas13-S-rev 5'-aatggcaggagcagttgtgaagttcttttc-3';
  • Cas12-N-rev 5'-gacttgatctttgaaatttggatctttg-3';
  • RNA fluorescent probe 5'-FAM-mArArUrGrGrGrCmAmArArUrGrGrGrCmA-BHQ1-3';
  • n represents the 2-position oxymethyl modification
  • r represents ribonucleotides
  • DNA fluorescent probe 5'-VIC-TTATTATT-BHQ1-3'.
  • the preferred technical scheme is: also includes bufferA solution and BufferB solution; bufferA solution configuration method is: add 50mmol of Tris buffer solution, 100nmol of potassium acetate, 20g of polyethylene glycol powder and 2mmol of dithiothreitol to 1L of water;
  • the BufferB solution is a magnesium acetate solution with a concentration of 280 mM.
  • the preferred technical scheme is: also includes: HEPES buffer solution, MgCl 2 solution, 10 ⁇ NEB buffer2.1 buffer solution, RNase inhibitor, T7 RNA polymerase, RNase-free water.
  • the technical solution provided by the present invention is: a double-target rapid detection method for novel coronavirus based on CRISPR/Cas system, comprising the following steps:
  • Step 1 Immerse the sample to be tested in the virus preservation solution, and then use the RNA extraction kit to extract the nucleic acid to obtain the nucleic acid to be tested;
  • Step 2 Add 37.5 ⁇ L of buffer A solution, 2 ⁇ L of Cas13-S-for, 2 ⁇ L of Cas13-S-rev, 2 ⁇ L of Cas12-N-for, 2 ⁇ L of Cas12-N-rev and 2 ⁇ L nucleic acid to be tested, then add 2.5 ⁇ L BufferB solution, cover the cap of the reaction tube and perform amplification reaction to obtain nucleic acid amplification product;
  • Cas13-S-for 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';
  • Cas13-S-rev 5'-aatggcaggagcagttgtgaagttcttttc-3';
  • Cas12-N-rev 5'-gacttgatctttgaaatttggatctttg-3';
  • Step 3 Prepare CRISPR reaction mixture: mix 0.4 ⁇ L of 1M HEPES buffer, 0.18 ⁇ L of 1M MgCl 2 solution, 1.6 ⁇ L of 10 ⁇ NEB buffer2.1 buffer, 0.8 ⁇ L of 25uM Each rNTPmix, 2 ⁇ L of LwaCas13a at a concentration of 63.2 ng/ ⁇ L, 1 ⁇ L of Cas13-crRNA at a concentration of 10 ng/ ⁇ l, 1 ⁇ L of LbaCas12a at a concentration of 1 uM, 1 ⁇ L of Cas12-crRNA at a concentration of 15 ng/ ⁇ l, 1 ⁇ L RNase inhibitor with a concentration of 40U/ ⁇ l, 0.1 ⁇ L of T7RNApolymerase with a concentration of 50U/ ⁇ l, 0.1 ⁇ L of a DNA fluorescent probe with a concentration of 100uM, 0.1 ⁇ L of an RNA fluorescent probe with a concentration of 100uM and 8.92 ⁇ L of RNA-free enzyme
  • n represents the 2-position oxymethyl modification
  • r represents ribonucleotides
  • Step 4 Add 2 ⁇ L of the nucleic acid amplification product obtained in step 2 to the CRISPR reaction mixture prepared in step 3, and incubate at 37°C for 30 minutes; judge by the following methods:
  • the fluorescence value of the FAM channel is greater than 3000, that is, the S site of the new coronavirus corresponding to Cas13a is detected positive, and the fluorescence value of the VIC channel is greater than 3000, that is, the corresponding site of Cas12a
  • the detection of the N site of the new coronavirus is positive, and the fluorescence value is less than 2000, which means that the corresponding site is negative. If the fluorescence value is between 2000-3000, it will be tested again. If the fluorescence value is still 2000-3000, it is determined that the corresponding site is positive.
  • bufferA solution configuration method is: add 50mmol of Tris buffer solution, 100nmol of potassium acetate, 20g of polyethylene glycol powder and 2mmol of dithiothreitol to 1L of water.
  • the present invention overcomes the incompatibility between the DETECTR and SHERLOCK systems, and successfully combines the LwaCas13a (Leptotrichia wadei Cas13a) derived from Leptotrichia wadei Cas13a with the Cas13a derived from Streptomyces ND2006 without reducing the sensitivity and accuracy of the detection method.
  • LbaCas12a (Lachnospiraceae bacterium ND2006 Cas12a) is unified to realize the simultaneous detection of two target sites using the CRISPR system.
  • Example 1 A novel coronavirus dual-target rapid detection method and kit based on CRISPR/Cas system
  • Kit includes:
  • Cas13-S-for 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';
  • Cas13-S-rev 5'-aatggcaggagcagttgtgaagttcttttc-3';
  • Cas12-N-rev 5'-gacttgatctttgaaatttggatctttg-3';
  • RNA fluorescent probe 5'-FAM-mArArUrGrGrGrCmAmArArUrGrGrGrCmA-BHQ1-3';
  • m 2-position oxygen methyl modification
  • r is RNA
  • DNA fluorescent probe 5'-VIC-TTATTATT-BHQ1-3';
  • BufferB solution BufferB solution, HEPES buffer, MgCl 2 solution, 10 ⁇ NEB buffer2.1 buffer, RNase inhibitor, T7 RNA polymerase and RNase-free water.
  • the bufferA solution configuration method is: add 50mmol Tris buffer solution, 100nmol potassium acetate, 20g polyethylene glycol powder and 2mmol dithiothreitol to 1L water; BufferB solution is the acetic acid whose concentration is 280mM Magnesium solution.
  • the process and principle of CRISPR-based dual-target detection are shown. First extract the nucleic acid of the sample to be tested, and then perform constant temperature amplification of the extracted nucleic acid at two sites (A/B site) by RT-RAA to amplify the signal to be detected, and the obtained constant temperature amplification product is added to the configured
  • LbaCas12a and LwaCas13a recognize their respective target nucleic acids under the guidance of their respective crRNAs and activate the corresponding non-specific cleavage capabilities.
  • crRNA is a guide RNA consisting of a fixed scaffold and a spacer complementary to the target sequence.
  • LbaCas12a recognizes the A site through Cas12acrRNA and activates the non-specific cleavage ability to cleave the surrounding single-stranded DNA probe to generate a fluorescent signal (VIC) to report the detection result of the A site.
  • LwaCas13a recognizes the B site through Cas13acrRNA and activates the non-specific cleavage ability to cleave the surrounding single-stranded RNA probe to generate a fluorescent signal (FAM) to report the detection result of the B site.
  • FAM fluorescent signal
  • a novel coronavirus dual-target rapid detection method based on the CRISPR/Cas system comprises the following technical steps.
  • Specimens should be extracted and tested for nucleic acid as soon as possible. Specimens that can be detected within 24 hours can be stored at 4°C; specimens that cannot be detected within 24 hours should be stored at -70°C or below. Nucleic acid extraction using the RNA extraction kit, with the kit Take the Mini Kit (QIAGEN, Cat No.
  • 74106 as an example: take 200 ⁇ L of virus preservation solution, add 350 ⁇ L of BufferRLT to mix by pipetting, and then add 550 ⁇ L of 70% absolute ethanol to precipitate viral RNA. The obtained turbid suspension was centrifuged by column filtration, 12000 rpm, 2 min, 4°C. Use BufferRW1 and BufferRPE successively to elute impurities, and finally add 80 ⁇ L of RNase-free water to the adsorption column, and dissolve and elute the viral nucleic acid by centrifugation.
  • RT-RAA reverse-Transcription-Recombinase-aid Amplification
  • reverse transcriptase reverse-transcribes RNA into cDNA, and then amplifies the target fragment at a constant temperature of 37°C under the guidance of multiple recombinases and specific RT-RAA primers.
  • the RT-RAA reaction is the first step in the CRISPR detection method, which can amplify the signal and improve the detection sensitivity.
  • RT-RAA primers follows the following principles: 1. The length of the primer is 30-35 bases; 2. The GC content of the primer is 30% ⁇ GC ⁇ 70%; 3. The length of the amplified product is between 100bp-200bp; 4. . The GC content of the amplified region needs to be 40% ⁇ GC ⁇ 60%, avoiding single repetitive sequences and palindromic sequences. Design a set of upstream and downstream primers around the detection site.
  • the present invention is a dual-target nucleic acid detection, it is necessary to design two sets of RT-RAA amplification primers, one set of RT-RAA primers for the N gene targeted by Cas12a, and one set of RT-RAA primers for the S gene targeted by Cas13a primers. Since Cas13a recognizes RNA, the DNA fragment obtained by constant temperature amplification needs to be transcribed into RNA, so the RT-RAA primer of Cas13a adds a section of T7 promoter recognition site (indicated by capital letters) at the 5' end of the upstream primer. RNA transcription during CRISPR detection.
  • Cas13-S-for 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';
  • Cas13-S-rev 5'-aatggcaggagcagttgtgaagttcttttc-3';
  • Cas12-N-rev 5'-gacttgatctttgaaatttggatctttg-3'.
  • RT-RAA amplification system For 50 ⁇ L RT-RAA amplification system, take the reaction tube containing protease freeze-dried powder, and add 37.5 ⁇ L buffer A solution (50mM Tris pH 7.9, 100nM Potassium acetate, 5% polyethylene glycol (PEG ), 2mM dithiothreitol (DTT)), 2 ⁇ L (10 ⁇ M) of each of the two target site RT-RAA upstream primers, 2 ⁇ L (10 ⁇ M) of each of the two target site RT-RAA downstream primers, and 2 ⁇ L of the nucleic acid to be tested.
  • buffer A solution 50mM Tris pH 7.9, 100nM Potassium acetate, 5% polyethylene glycol (PEG ), 2mM dithiothreitol (DTT)
  • 2 ⁇ L (10 ⁇ M) of each of the two target site RT-RAA upstream primers 2 ⁇ L (10 ⁇ M) of each of the two
  • Buffer B solution 280 mM magnesium acetate solution (Magnesium Acetate)
  • the detection of LbaCas12a requires a PAM region for recognition on the sequence to be recognized, and the sequence of the PAM region is TTTN.
  • the crRNA design of LwaCas13a has no special requirements. Therefore, by comparing the genome sequence of the new coronavirus (NC_045512) with several other coronaviruses, including hCoV-229E (NC_002645), hCoV-HKU1 (NC_006577), hCoV-NL63 (NC_005831), hCoV-OC43 (NC_006213), SARS- CoV (NC_004718) and bat SARS-like coronavirus (MG772933), avoiding homologous sequences, finally designed detection sites for LbaCas12a and LwaCas13a.
  • the detection site of final design of the present invention is as follows:
  • the present invention finally succeeded in combining LbaCas12a and LwaCas13a by adding different ions. It is integrated and can be used for rapid ultrasensitive dual-target detection of the new coronavirus.
  • the CRISPR reaction mixture configuration system is as follows:
  • RNA fluorescent probe (m is 2-position oxymethyl modification, r is RNA);
  • VOC-DNA probe DNA fluorescent probe
  • the qPCR instrument Bio-Rad CFX96
  • the qPCR instrument can be used to read the fluorescence values under the FAM and VIC channels, incubate at 37°C for 20 cycles, with an interval of 2.5 minutes between each cycle, and record the fluorescence signal once at the end of each cycle. Judging the result of dual-target detection by the final fluorescence signal intensity,
  • a fluorescence value greater than 3000 indicates that the corresponding site is detected positively. If the fluorescence value of the FAM channel is greater than 3000, it means that the S site of the new coronavirus corresponding to Cas13a is detected positive.
  • the fluorescence value of the VIC channel is greater than 3000, that is, the N site of the new coronavirus corresponding to Cas12a is detected positive. If the fluorescence value is less than 2000, it means that the detection of the corresponding site is negative. If the fluorescence value is between 2000-3000, it will be tested again. If the fluorescence value is still 2000-3000, it will be determined that the detection of the corresponding site is positive.
  • the FAM channel is a fluorescence detection channel when the qPCR instrument reads fluorescence, and reads fluorescence signals with a wavelength of 450nm-490nm.
  • the VIC channel reads at a wavelength of 500-535nm.
  • RT-RAA amplification primers, crRNA and single-stranded probes were synthesized by Nanjing Qingke Biotechnology Co., Ltd. and Nanjing GenScript Biotechnology Co., Ltd.
  • RT-RAA nucleic acid basic amplification kit was purchased from Hangzhou Zhongce Biological Company.
  • LbaCas12a protein was purchased from NEB.
  • LwaCas13a protein was purchased from Nanjing GenScript Biotechnology Co., Ltd.
  • LbaCas12a requires a PAM region for recognition on the sequence to be recognized, and the sequence of the PAM region is TTTN.
  • the crRNA design of LwaCas13a has no special requirements.
  • the final designed crRNA detection site of the present invention is as follows:
  • the virus genome is first amplified by RT-RAA at constant temperature, and then added to the prepared multi-group CRISPR dual-target reaction system, and the optimal reaction system is judged by monitoring the fluorescence signal of the FAM/VIC channel. That is, under a fixed virus concentration, the monitoring result has the highest fluorescence value and the most stable combination of monitoring results. It is known from experiments that when 80% of the LbaCas12a reaction system is introduced, the efficiency and stability of dual-target detection are the best. Both sites were efficiently detected. As shown in Figure 4
  • Metal ions are prosthetic groups or activators of enzymes, which can help to stabilize the conformation, constitute the active center of the enzyme, or act as a link, serving as a bridge to integrate the enzyme and the substrate.
  • Common enzymatic divalent metal ions include Mg, Ca, Mn, Ni, Zn, Co, Cu, etc.
  • the present invention screens divalent ions such as Mg, Ca, Mn, and Ni, and finally obtains the most suitable metal ion and its concentration for the CRISPR dual-target detection method. In this example, under the buffer system that has been explored, by setting a single variable, the influence of different metal ions on the detection of dual-target CRISPR is verified.
  • novel coronavirus CRISPR dual-target detection method in the present invention can stably detect the sensitivities of the two sites: Cas13a 7cp/ul, Cas12a 25cp/ul.
  • Figure 6 shows that the novel coronavirus CRISPR dual-target detection method in the present invention can stably detect the sensitivities of the two sites: Cas13a 7cp/ul, Cas12a 25cp/ul.
  • the sample is first processed to obtain nucleic acid, and then two pairs of RT-RAA primers are used to simultaneously perform constant temperature amplification on the two detected nucleic acid sites, and finally the obtained RT-RAA product is added to the prepared dual-target CRISPR reaction system , incubate at 37°C for one hour and detect the fluorescent signals of the FAM and VIC channels to obtain the detection results.
  • the present invention also performs homologous alignment of the sequence of SARS-CoV-2 with various other coronaviruses, including human coronavirus 229E, HKU1, NL63 , OC43, SARS-CoV and bat SARS-CoV.
  • the blue part in the figure is the aligned homologous sequence, and the red part is the difference sequence between the genomes.
  • different ratios of Cas12a protein reaction system were introduced into the reaction system of Cas13a protein, namely 100%, 80%, 60%, 40% and 20%.
  • Use a fixed concentration of the new coronavirus template (10000cp/ul) first undergo dual-site RT-RAA constant temperature amplification, and then take 2ul volumes of RT-RAA amplification products and add them to the CRISPR dual-target detection system with different component ratios , incubate at 37°C for 1 hour, and detect the fluorescence values of the FAM and VIC channels every 2 minutes throughout the incubation.
  • LwaCas13a cuts the FAM-labeled RNA probe
  • LbaCas12a cuts the VIC-labeled DNA probe
  • FAM positive means that the gene locus detected by lwaCas13a is positive
  • VIC positive means that the gene locus detected by LbaCas12a is positive.
  • the complete CRISPR dual-target detection method is used to detect the diluted templates at different concentrations to verify the sensitivity of the detection method. It can be seen from the figure that the novel coronavirus CRISPR dual-target detection method in the present invention can stably detect the sensitivities of the two sites: Cas13a 7cp/ul, Cas12a 25cp/ul.
  • Example 2 A novel coronavirus dual-target rapid detection method and kit based on CRISPR/Cas system
  • kits for rapid detection of novel coronavirus double targets based on CRISPR/Cas system comprising:
  • Cas13-S-for 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';
  • Cas13-S-rev 5'-aatggcaggagcagttgtgaagttcttttc-3';
  • Cas12-N-rev 5'-gacttgatctttgaaatttggatctttg-3';
  • RNA fluorescent probe 5'-FAM-mArArUrGrGrGrCmAmArArUrGrGrGrCmA-BHQ1-3';
  • n represents the 2-position oxymethyl modification
  • r represents ribonucleotides
  • DNA fluorescent probe 5'-VIC-TTATTATT-BHQ1-3'.
  • bufferA solution also includes bufferA solution and BufferB solution;
  • bufferA solution configuration method is: add 50mmol Tris buffer solution, 100nmol potassium acetate, 20g polyethylene glycol powder and 2mmol dithiothreitol to 1L water;
  • BufferB solution is 280mM magnesium acetate solution.
  • HEPES buffer MgCl 2 solution
  • 10 ⁇ NEB buffer2.1 buffer 10 ⁇ NEB buffer2.1 buffer
  • RNase inhibitor T7 RNA polymerase
  • RNase-free water also includes: HEPES buffer, MgCl 2 solution, 10 ⁇ NEB buffer2.1 buffer, RNase inhibitor, T7 RNA polymerase, RNase-free water.
  • a novel coronavirus dual-target rapid detection method based on CRISPR/Cas system comprising the following steps:
  • Step 1 Immerse the sample to be tested in the virus preservation solution, and then use the RNA extraction kit to extract the nucleic acid to obtain the nucleic acid to be tested; the sample to be tested is extracted from the doorknob.
  • Step 2 Add 37.5 ⁇ L of bufferA solution, 2 ⁇ L of Cas13-S-for, 2 ⁇ L of Cas13-S-rev, 2 ⁇ L of Cas12-N-for, 2 ⁇ L of Cas12 into a reaction tube containing protease lyophilized powder -N-rev and 2 ⁇ L of the nucleic acid to be tested, then add 2.5 ⁇ L of BufferB solution, cover the cap of the reaction tube and perform the amplification reaction to obtain the nucleic acid amplification product;
  • Cas13-S-for 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';
  • Cas13-S-rev 5'-aatggcaggagcagttgtgaagttcttttc-3';
  • Cas12-N-rev 5'-gacttgatctttgaaatttggatctttg-3';
  • Step 3 Prepare CRISPR reaction mixture: mix 0.4 ⁇ L of 1M HEPES buffer, 0.18 ⁇ L of 1M MgCl2 solution, 1.6 ⁇ L of 10 ⁇ NEB buffer2.1 buffer, 0.8 ⁇ L of 25uM per rNTPmix, 2 ⁇ L of LwaCas13a at a concentration of 63.2ng/ ⁇ L, 1 ⁇ L of Cas13-crRNA at a concentration of 10ng/ ⁇ l, 1 ⁇ L of LbaCas12a at a concentration of 1uM, 1 ⁇ L of a concentration of 15ng/ ⁇ l Cas12-crRNA, 1 ⁇ L of RNase inhibitor at a concentration of 40U/ ⁇ l, 0.1 ⁇ L of T7RNApolymerase at a concentration of 50U/ ⁇ l, 0.1 ⁇ L of a DNA fluorescent probe at a concentration of 100uM, 0.1 ⁇ L of an RNA fluorescent probe at a concentration of 100uM, and 8.92 ⁇ L of RNase-free water mix;
  • n represents the 2-position oxymethyl modification
  • r represents ribonucleotides
  • Step 4 Add 2 ⁇ L of the nucleic acid amplification product obtained in step 2 to the CRISPR reaction mixture prepared in step 3, and incubate at 37°C for 30 minutes; judge by the following methods:
  • the fluorescence value of the FAM channel is greater than 3000, that is, the S site of the new coronavirus corresponding to Cas13a is detected positive, and the fluorescence value of the VIC channel is greater than 3000, that is, the corresponding site of Cas12a
  • the N site of the new coronavirus is positive, and the fluorescence value is less than 2000, which means that the corresponding site is negative. If the fluorescence value is between 2000-3000, re-test. If the fluorescence value is still 2000-3000, the corresponding site is determined to be positive.

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Abstract

Procédé et kit de détection rapide à double cible du SARS-CoV-2 reposant sur le système CRISPR/Cas. Le procédé comprend les étapes suivantes : réalisation d'une extraction d'acide nucléique pour obtenir un acide nucléique à détecter ; amplification simultanée de deux sites à détecter en utilisant deux groupes d'amorces RT-RAA pour obtenir des produits d'acide nucléique, à savoir un produit d'amplification du gène N ciblé par Cas12a et un produit d'amplification du gène S ciblé par Cas13a ; et préparation d'une solution mixte de réaction CRISPR, ajout de 2 μL d'un produit d'amplification d'acide nucléique dans la solution mixte de réaction CRISPR, incubation à 37 °C pendant 30 min, et lecture de la fluorescence pour obtenir un résultat de détection.
PCT/CN2022/091168 2021-06-10 2022-05-06 Procédé et kit de détection rapide de sars-cov-2 à base de système crispr/cas WO2022257659A1 (fr)

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CN116949218A (zh) * 2023-08-04 2023-10-27 中国水产科学研究院珠江水产研究所 用于检测ⅲ型鲤疱疹病毒的raa-crispr试剂盒
CN117305484A (zh) * 2023-10-25 2023-12-29 四川省畜牧科学研究院 一种同时检测多杀性巴氏杆菌和溶血性曼氏杆菌的检测体系和方法

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