WO2022257663A1 - Procédé et kit pour la détection et le dépistage de la mutation n501y dans la covid-19 - Google Patents

Procédé et kit pour la détection et le dépistage de la mutation n501y dans la covid-19 Download PDF

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WO2022257663A1
WO2022257663A1 PCT/CN2022/091331 CN2022091331W WO2022257663A1 WO 2022257663 A1 WO2022257663 A1 WO 2022257663A1 CN 2022091331 W CN2022091331 W CN 2022091331W WO 2022257663 A1 WO2022257663 A1 WO 2022257663A1
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solution
mutation
fluorescence
raa
nucleic acid
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杜忆南
邢晨
柳燕
祝亚亭
李倩
王小凤
张智康
张宁
王悦晨
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安徽医科大学
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/701—Specific hybridization probes
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    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the technical field of biological detection, and relates to a method and a kit for detecting and screening the N501Y mutation of the new coronavirus.
  • 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 coronavirus S protein spike protein
  • the new coronavirus recognizes the hACE2 receptor on the surface of the host cell through the surface protein to invade the cell.
  • the human body also realizes virus immunity by recognizing virus surface antigens and producing corresponding antibodies.
  • Most of the current new crown vaccines are also designed for the S protein of the new crown virus.
  • N501Y mutation is a base change from A to T at the first base of amino acid No. 501 of the new coronavirus S protein, resulting in the amino acid being changed from asparagine (N) to tyrosine (Y).
  • N asparagine
  • Y tyrosine
  • Single nucleotide polymorphism refers to the polymorphism of nucleic acid sequence caused by the change of a single nucleotide base.
  • Single nucleotide polymorphism detection is a detection technology used to detect DNA sequence polymorphisms caused by single nucleotide variation at the genome level.
  • SNP detection methods are roughly divided into three categories: 1Gel-based detection of known polymorphisms, including polymerase chain reaction, restriction fragment length polymorphism markers, oligonucleotide ligation analysis, and small sequencing; Gel high-throughput detection technology, including fluorescence energy resonance transfer detection, mass spectrometry, and DNA chips; 3 conformation-based detection of unknown mutations, including single-strand conformational polymorphism, chemical or enzyme mismatch modification analysis, Denaturing gradient gel electrophoresis, denaturing high performance liquid chromatography, etc.
  • 1Gel-based detection of known polymorphisms including polymerase chain reaction, restriction fragment length polymorphism markers, oligonucleotide ligation analysis, and small sequencing
  • Gel high-throughput detection technology including fluorescence energy resonance transfer detection, mass spectrometry, and DNA chips
  • 3 conformation-based detection of unknown mutations including single-strand conformational polymorphism, chemical or enzyme mismatch modification analysis, Denaturing gradient gel electrophor
  • 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 of the CRISPR community which relies on A nucleic acid constant temperature detection technology called SHERLOCK for Cas13a.
  • the principle is to amplify and enrich the target fragment through a constant temperature amplification method such as RT-RAA, and then the Cas protein will target and recognize the amplified target fragment under the guidance of crRNA and be activated.
  • the activated Cas protein becomes a DNA cutter (Cas12a) or RNA cutter (Cas13a), 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 cleavage detection of the CRISPR/Cas system requires that the CRISPR/Cas protein binds to crRNA and recognizes and binds to the sequence to be detected under the guidance of crRNA, thereby activating the Cas protein for cleavage detection.
  • crRNA is a guide RNA consisting of a fixed scaffold and a spacer complementary to the target sequence.
  • Single-base mutation (SNP) detection uses the base recognition specificity of crRNA. By placing the mutant bases in different positions of the crRNA or artificially introducing base mismatches, the crRNA can only recognize the mutant sequence but not the sequence without SNP. The original sequence, so as to realize mutation typing detection.
  • LwaCas13a LeptotriciawadeiCas13a was used to distinguish and identify the N501Y mutation of the new coronavirus.
  • LwaCas13a can tolerate one base mismatch and activate for cleavage detection when recognizing crRNA, but LwaCas13a cannot recognize and activate in the case of two base mismatches.
  • the critical point of SNP detection can be reached by artificially introducing an additional base mismatch on the crRNA of LwaCas13a: when detecting the SNP sequence, there is only one artificial mismatch, and the crRNA activates the Cas protein for cleavage detection; when detecting SNP-free In the original sequence, because there are two base mismatches, the crRNA cannot activate the Cas protein to cause corresponding spatial structure changes, showing a negative test.
  • the crRNA sequence can detect the N501Y mutation most specifically and sensitively.
  • the purpose of the present invention is to provide a method and kit for detecting and screening the N501Y mutation of the new coronavirus.
  • kits for detecting and screening the N501Y mutation of the new coronavirus comprising:
  • a set of RT-RAA amplification primers :
  • RT-RAA downstream primer 5'-gttcaaaagaaagtactactactctgtatgg-3';
  • the preferred technical scheme is: also include bufferA solution and BufferB solution; buffer A solution configuration method is: add 50mmol Tris buffer solution, 100nmol potassium acetate, 20g polyethylene glycol powder and 2mmol dithiothreitol to 1L 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 RNApolymerase, RNase-free water.
  • the technical solution provided by the present invention is: a method for detecting and screening the N501Y mutation of the new coronavirus, 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 41.5 ⁇ L of bufferA solution, 2 ⁇ L of RT-RAA upstream primer, 2 ⁇ L of RT-RAA downstream primer and 2 ⁇ L of the nucleic acid to be tested to a reaction tube containing protease lyophilized powder, then add 2.5 ⁇ L of BufferB solution, and cover Carry out the amplification reaction behind the cap of the reaction tube to obtain the nucleic acid amplification product;
  • RT-RAA downstream primer 5'-gttcaaaagaaagtactactactctgtatgg-3';
  • Step 3 Prepare CRISPR reaction mixture: 0.4 ⁇ L of 1M HEPES buffer, 0.18 ⁇ L of 1M MgCl 2 solution, 0.8 ⁇ L of 10 uM rNTP mix, 2 ⁇ L of 63.2 ng/ ⁇ L of LwaCas13a, 1 ⁇ L of RNase inhibitor at a concentration of 40 U/ ⁇ L, 0.1 ⁇ L of T7 RNA polymerase at a concentration of 50 U/ ⁇ L, 0.5 ⁇ L of M1 crRNA at a concentration of 10 ng/ ⁇ L, and 0.2 ⁇ L of an RNA fluorescent probe at a concentration of 100 uM Mix the needle with 12.82 ⁇ L of RNase-free water;
  • 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 is still 2000-3000, it is determined that the N501Y test is positive; If there is no fluorescence reading instrument such as qPCR on site, you can also place the reaction tube under a transmitted light source with an excitation wavelength of 485nm after the reaction is completed to observe the color change to determine the test result; the reaction tube with a positive N501Y test will emit yellow-green fluorescence, and the N501Y test Negative tubes have no fluorescence.
  • 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 invention has the advantages of fast detection speed, high accuracy and low cost.
  • FIG 1 Schematic diagram of the principle of the mode of distinguishing SNPs by LwaCas13a.
  • Figure 2 Screening diagram of the best primers for RT-RAA at the N501Y site.
  • Fig. 4 crRNA screening diagram of N501Y Cas13a.
  • Example 1 A method and kit for detecting and screening the N501Y mutation of the new coronavirus
  • a method for detecting and screening the N501Y mutation of the new coronavirus comprising the following technical steps.
  • RNA extraction kit Take the Mini Kit (QIAGEN, CatNo.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. It is recommended to design 3 sets of RT-RAA primers each upstream and downstream around the detection site, and use the complete CRISPR detection cleavage reaction to screen out the RT-RAA primers with the best amplification effect.
  • the present invention designs 3 sets of primers for the mutation of the novel coronavirus N501Y, and after final screening, the best amplification primers are obtained as follows:
  • Cas13-501-for2 5'-GAAATTAATACGACTCACTATAGGGccttgtaatggtgttgaaggttttaattgttac-3';
  • Cas13-501-rev1 5'-gttcaaaagaaagtactactactctgtatgg-3';
  • RT-RAA amplification system For 50 ⁇ L RT-RAA amplification system, take the reaction tube containing protease freeze-dried powder, and add 41.5 ⁇ L buffer A solution (50mM Tris pH 7.9, 100nM Potassium acetate, 5% polyethylene glycol (PEG ), 2mM dithiothreitol (DTT)), 2 ⁇ L RT-RAA upstream primer (Cas13-501-for2) (10 ⁇ M), 2 ⁇ L RT-RAA downstream primer (Cas13-501-rev1) (10 ⁇ M) and 2 ⁇ L to be tested nucleic acid.
  • buffer A solution 50mM Tris pH 7.9, 100nM Potassium acetate, 5% polyethylene glycol (PEG ), 2mM dithiothreitol (DTT)
  • 2 ⁇ L RT-RAA upstream primer Cas13-501-for2
  • 2 ⁇ L RT-RAA downstream primer Cas13-501-rev1
  • Buffer B solution 280 mM magnesium acetate solution (Magnesium Acetate)
  • a total of 11 crRNAs of LwaCas13a were designed for the identification and identification of the N501Y mutation of the new coronavirus.
  • the artificial mismatched bases are placed at the 2nd, 3rd, 4th, 5th, and 6th bases of the crRNA corresponding to the spacer, and are designed in combination.
  • the crRNA sites designed above were screened for complete fluorescent cutting detection.
  • the present invention finally screened out a specific and sensitive crRNA sequence that can be used to identify and distinguish the N501Y mutation, which is now announced as follows:
  • the N501Y mutation was detected using the CRISPR/Cas13a system.
  • the CRISPRCas13a reaction mixture configuration system is as follows:
  • the effect of the CRISPR reaction system on SNP detection is mainly reflected in the specificity, that is, the specific base recognition characteristics of the CRISPR system endow it with the ability to detect SNP.
  • the crRNA provided by the present invention can effectively recognize the N501Y mutation sequence and activate the corresponding Cas protein, and cleave the surrounding fluorescent reporter probe to show that the N501Y mutation is detected positively.
  • the crRNA detects the original sequence without mutations the corresponding Cas protein cannot be activated due to base mismatches, thus showing that the N501Y mutation is negative.
  • the cycle interval was 2 minutes, and the fluorescence signal was recorded once at the end of each cycle. Judging the N501Y test result by the final fluorescence signal intensity, that is, the fluorescence value greater than 3000 means that the N501Y test is positive, and the fluorescence value is less than 2000 means that the N501Y test is negative. 3000 means that the N501Y test is positive. If there is no fluorescent reading instrument such as qPCR on site, the reaction tube can also be placed under a transmitted light source with an excitation wavelength of 485nm after the reaction is completed to observe the color change to determine the detection result. N501Y-positive reaction tubes will emit yellow-green fluorescence, while N501Y-negative reaction tubes will have no fluorescence.
  • Example 2 A method and kit for detecting and screening the N501Y mutation of the new coronavirus
  • 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.
  • LwaCas13a protein was purchased from Nanjing GenScript Biotechnology Co., Ltd.
  • the pseudovirus containing the mutated sequence is packaged in the laboratory, and a company can also be entrusted to package the pseudovirus containing the corresponding mutated sequence.
  • RT-RAA amplification primers for the N501Y mutation site were designed. According to the design requirements of RT-RAA, three upstream primers and three downstream primers were designed, and the specific sequences are as follows:
  • a primer combination with the highest amplification efficiency was screened through the complete RT-RAA-CRISPRCas13a experiment.
  • a Cas13a was designed to detect the crRNA at the original site of 501N. The sequence is as follows:
  • the amplification efficiency of each set of primers was verified using different concentration gradients of the S fragment RNA template of the new coronavirus, and the molecular weight was calculated according to the length of the RNA template.
  • the molecular copy number converted into a 50ng/ ⁇ L RNA template was about 1.4 ⁇ 1011cp/ ⁇ L. It was diluted 10-fold to 1400cp/ ⁇ L, 140cp/ ⁇ L, 14cp/ ⁇ L, 1.4cp/ ⁇ L, 0.7cp/ ⁇ L and 0cp/ ⁇ L.
  • RT-RAA-CRISPRCas13 cutting detection to verify the amplification efficiency of a total of 9 primer combinations according to the above 6 concentration gradients, judge the amplification efficiency of the primers by the fluorescence intensity, and finally select a set of optimal amplification primers .
  • the N501Y mutation causes the first base of amino acid 501 of the new coronavirus S protein to be mutated from adenine (A) to thymine (T).
  • To detect the N501Y mutation is to detect the change of the base.
  • LwaCas13a is used to detect the mutation from N501Y adenine (A) to thymine (T).
  • crRNA is a guide RNA consisting of a fixed scaffold and a spacer complementary to the target sequence. According to existing reports, Cas13a can tolerate a base mismatch on the spacer and be successfully activated. Therefore, in the detection When N501Y is mutated, in the case of a mutation mismatch, we introduce an additional artificial mismatch on the spacer, that is, it is possible to make crRNA only recognize the mutant sequence and not the original sequence, so as to achieve the purpose of mutation typing. After a comprehensive analysis of the spatial structure of the Cas13a protein interacting with crRNA, the present invention artificially introduced base mismatches at the 3rd, 4th, 5th, and 6th bases of the spacer, and designed a total of 11 crRNAs for subsequent screening. The specific sequence is shown in the table below, and the sequence display is shown in Figure 3.
  • a pseudovirus containing the N501Y mutant sequence was used to simulate the positive sample of the virus mutation, and a pseudovirus containing the original sequence was used as a negative control.
  • the 11 crRNAs designed above were screened with 100 cp/ ⁇ L of the two pseudovirus samples.
  • the Cas13acrRNA that can detect the N501Y mutation but cannot detect the 501N original sequence was screened by the fluorescence intensity detected by RT-RAA-CRISPR Cas13a cleavage.
  • the fluorescence intensity of different primer sets was compared through the complete RT-RAA-CRISPRCas13a cleavage detection reaction, and the fluorescence signal was collected by Bio-Rad CFX96.
  • the combination of primer F2+R1 (Cas13-501-for2+Cas13-501-rev1) has the highest detection sensitivity, can stably detect viral nucleic acid with a concentration of 0.7cp// ⁇ L, and has the highest primer amplification efficiency, so This combination was selected as the best RT-RAA amplification primer.
  • Effective crRNA was screened by RT-RAA-CRISPRCas13a cleavage detection reaction using mutation-containing positive samples and mutation-free negative samples.
  • the concentration of the pseudovirus sample was 100cp/ ⁇ L, two repetitions were set, and the nucleic acid was extracted using a nucleic acid extraction kit. And use Zhongce RT-RAA nucleic acid basic amplification kit for constant temperature amplification.
  • Prepare a separate CRISPR reaction system for each crRNA take 18 ⁇ L CRISPR reaction system and add 2 ⁇ L RT-RAA amplification product, incubate at 37°C for 20 cycles, and collect fluorescence through Bio-Rad CFX96 throughout the incubation.
  • the mutation discrimination ability of each crRNA was judged by comparing the fluorescence intensity between the mutation sample and the original sample. As shown in Figure 4, Cas13acrRNAM1 can effectively detect the N501Y mutation, but not the original template.
  • Figure 5 shows that crRNAM1 can effectively detect the N501Y-G mutation at 1400cp/ ⁇ L, 140cp/ ⁇ L, 14cp/ ⁇ L, 1.4cp/ ⁇ L and 0.7cp/ ⁇ L, and the corresponding concentration of the original template showed negative detection.
  • the reaction tube was placed under a 485nm transmitted light source, and it was observed that the reaction tube of the mutant template emitted obvious yellow-green fluorescence, while the reaction well of the original template had no fluorescence.
  • Figure 1 Demonstrates the principle of mutation detection using CRISPR/Cas13a. That is, the mutant strain is more compatible with the guide RNA (crRNA), thereby activating the Cas protein for cleavage detection.
  • crRNA guide RNA
  • Figure 2 Shows the screening results of RT-RAA primers at the N501Y site. There are 9 combinations of the three sets of upstream and downstream primers paired in pairs, and the figure shows the fluorescence intensity of each primer combination when detecting virus templates with different concentration gradients. Screening results showed that primer F2+R1 had the best amplification effect.
  • Figure 3 The base sequence diagram shows the 11 crRNAs designed for LwaCas13a to distinguish the N501Y mutation.
  • the Cas effector protein can be recognized and activated when detecting mutant strains, but cannot be recognized when detecting original strains.
  • Figure 4 Shows the screening effects of various crRNA detection mutations, using each designed crRNA to detect N501Y mutation samples and original strain samples, and comparing the mutation detection effects. Screening results showed that M1crRNA had the strongest ability to distinguish mutations.
  • FIG. 5 Further validation results for the screened M1crRNA. Using M1crRNA and CRISPR/Cas13a system to detect mutant strain samples with different concentration gradients and original strain samples, it was shown that M1crRNA can achieve good mutation detection results.
  • Figure 6 Demonstrates direct readout of assay results without relying on a fluorescence reading machine. Place the reaction tube under 485nm excitation light, and the yellow-green fluorescence of the mutant group can be observed with the naked eye.
  • Example 3 A method and kit for detecting and screening the N501Y mutation of the new coronavirus
  • kits for detecting and screening the N501Y mutation of the new coronavirus comprising:
  • a set of RT-RAA amplification primers :
  • RT-RAA downstream primer 5'-gttcaaaagaaagtactactactctgtatgg-3';
  • bufferA solution also include bufferA solution and BufferB solution;
  • buffer A solution configuration method is: add 50mmol Tris buffer solution, 100nmol potassium acetate, 20g polyethylene glycol powder and 2mmol dithiothreitol to 1L water; BufferB solution has a concentration of 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 method for detecting and screening the N501Y mutation of the new coronavirus 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 nucleic acid to obtain the nucleic acid to be tested; the sample to be tested is extracted from the doorknob.
  • Step 2 Add 41.5 ⁇ L of buffer A solution, 2 ⁇ L of RT-RAA upstream primer, 2 ⁇ L of RT-RAA downstream primer and 2 ⁇ L of nucleic acid to be tested to a reaction tube containing protease lyophilized powder, and then add 2.5 ⁇ L of BufferB solution , after covering the cap of the reaction tube, the amplification reaction is carried out to obtain the nucleic acid amplification product;
  • RT-RAA downstream primer 5'-gttcaaaagaaagtactactactctgtatgg-3';
  • Step 3 Prepare CRISPR reaction mixture: 0.4 ⁇ L of 1M HEPES buffer, 0.18 ⁇ L of 1M MgCl 2 solution, 0.8 ⁇ L of 10 uM rNTP mix, 2 ⁇ L of 63.2 ng/ ⁇ L of LwaCas13a, 1 ⁇ L of RNase inhibitor at a concentration of 40 U/ ⁇ L, 0.1 ⁇ L of T7 RNA polymerase at a concentration of 50 U/ ⁇ L, 0.5 ⁇ L of M1 crRNA at a concentration of 10 ng/ ⁇ L, and 0.2 ⁇ L of an RNA fluorescent probe at a concentration of 100 uM Mix the needle with 12.82 ⁇ L of RNase-free water;
  • 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 is still 2000-3000, it is determined that the N501Y test is positive; If there is no fluorescence reading instrument such as qPCR on site, you can also place the reaction tube under a transmitted light source with an excitation wavelength of 485nm after the reaction is completed to observe the color change to determine the test result; the reaction tube with a positive N501Y test will emit yellow-green fluorescence, and the N501Y test Negative reaction tubes have no fluorescence.

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Abstract

Procédé et kit pour la détection et le dépistage de la mutation N501Y dans la covid-19. Le procédé comprend la réalisation d'une extraction d'acide nucléique pour obtenir un acide nucléique à tester, l'utilisation d'une amorce conçue pour la réaction d'amplification pour obtenir un produit d'amplification d'acide nucléique, la préparation d'un mélange réactionnel CRISPR, l'ajout de 2 μL de produit d'amplification d'acide nucléique au mélange réactionnel CRISPR, l'incubation à 37 °C pendant 30 minutes, et la lecture d'un résultat de détection par fluorescence.
PCT/CN2022/091331 2021-06-10 2022-05-07 Procédé et kit pour la détection et le dépistage de la mutation n501y dans la covid-19 WO2022257663A1 (fr)

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