CN113881806A - Method and kit for detecting novel coronavirus and 69/70 mutant strain based on CRISPR/Cas12a technology - Google Patents
Method and kit for detecting novel coronavirus and 69/70 mutant strain based on CRISPR/Cas12a technology Download PDFInfo
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Abstract
The invention discloses a method and a kit for detecting a novel coronavirus and a 69/70 mutant strain based on a CRISPR/Cas12a technology. According to the invention, through the capacity of specifically recognizing the DNA target by the LbaCas12a/crRNA system, the 69/70-crRNA-W and 69/70-crRNA-M which are deleted aiming at the non-mutated target point 69/70 and the mutated target point 69/70 of the new coronavirus are respectively designed, the sample molecules are detected, only one PCR instrument or a constant temperature device and a simple fluorescence reading device are needed, when two crRNAs are used for detecting the same sample, the wild type or 69/70 deletion mutant strain of the new coronavirus in a nucleic acid sample can be rapidly and effectively distinguished, and the method has the characteristics of high specificity, high sensitivity and low cost, and is more widely suitable for various molecular diagnosis laboratories. The invention has important significance for the prevention and control of the new coronavirus epidemic situation.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a method and a kit for rapidly detecting wild type and mutant strain (69/70 deletion mutation) of a new coronavirus (SARS-CoV-2), and specifically, the invention utilizes the specificity of Cas12a/crRNA to rapidly detect the 69/70 deletion mutation of the wild type and the mutant strain of SARS-CoV-2.
Background
The SARS-CoV-2 mutation gradually increased. Among the B.1.1.7 mutants found in the UK, there is an 69/70 deletion mutation resulting from the deletion of amino acids 69 and 70 in the S protein of the novel coronavirus (SARS-CoV-2), which deletion may double the infectivity of the virus and thus exacerbate the difficulty of controlling epidemic situations.
The detection method of the new coronavirus is mainly divided into a nucleic acid detection method and an antibody detection method, and because of the requirements of sensitivity and specificity, the detection of the mutation of the new coronavirus is mainly performed by a Whole Genome Sequencing (WGS) or Sanger sequencing method, but the detection method is slow and is not suitable for all molecular diagnosis laboratories. Therefore, the development of a rapid, sensitive, convenient and low-cost detection method and kit for the deletion mutation of the novel coronavirus 69/70 is of great significance to epidemic prevention and treatment and early discovery of novel crown mutation strains in the field.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary object of the present invention is to provide a kit for rapidly detecting wild type and mutant strains of a novel coronavirus based on CRISPR/Cas12a technology.
Another object of the present invention is to provide a method for rapidly detecting new coronavirus wild type and mutant strain based on CRISPR/Cas12a technology. The method is a method for detecting the wild type and 69/70 deletion mutant strains of the novel coronavirus by utilizing an LbaCas12a/crRNA system.
The purpose of the invention is realized by the following technical scheme:
a kit for rapidly detecting wild type and mutant strains of a new coronavirus based on CRISPR/Cas12a technology comprises LbaCas12a, a wild-type specific 69/70-crRNA-W sequence aiming at 69/70 locus, a specific 69/70-crRNA-M sequence aiming at 69/70 deletion mutant strains, RT-RAA primers or RT-PCR primers aiming at wild-type and mutant strain characteristic sequences of the new coronavirus, and a report single-stranded DNA molecule;
in order to better realize the invention, the kit can be combined with RT-RAA, RT-RPA and other isothermal amplification technologies or reverse transcription PCR (RT-PCR) technologies to carry out reverse transcription and amplification on the novel coronavirus nucleic acid;
preferably, the kit also comprises A Buffer, NEBuffer2.1, RNase Inhibitor, freeze-dried RT-RAA reaction microspheres and MgAc.
The specific 69/70-crRNA-W sequence is shown as SEQ ID NO: 1, the sequence of the specificity 69/70-crRNA-M is shown as SEQ ID NO: 2, unlike the conventional crRNA design, the PAM sequence next to this sequence is TTC instead of conventional TTTV.
The RT-RAA primers aiming at the wild type and mutant strain characteristic sequences of the new coronavirus are 69/70-RT-RAA-F/R primer groups (SEQ ID NO: 3-4), and the RT-RAA primers are high-efficiency specific sequences screened by comparing high-throughput sequences of the new coronavirus, analyzing the mutation frequency of a primer candidate region and combining the molecular epidemiology characteristics of the new coronavirus and the requirements of the RT-RAA primers;
the RT-PCR primers aiming at the wild type and mutant strain characteristic sequences of the new coronavirus are 69/70-RT-PCR-F/R primer groups (SEQ ID NO: 5-6), and the RT-PCR primers are high-efficiency specific sequences screened by comparing high-throughput sequences of the new coronavirus, analyzing the mutation frequency of a primer candidate region and combining the molecular epidemiology characteristics of the new coronavirus and the requirements of the RT-PCR primers;
the 69/70-RT-RAA-F/R primer group is used for amplifying a nucleic acid fragment containing complementarity with 69/70-crRNA-W and/or 69/70-crRNA-M;
the 69/70-RT-PCR-F/R primer group is used for amplifying a nucleic acid fragment containing complementarity with 69/70-crRNA-W and/or 69/70-crRNA-M;
the report single-stranded DNA molecule is SEQ ID NO: 7 or SEQ ID NO: 8.
a method for rapid detection of new coronavirus wild-type and mutant strains based on CRISPR/Cas12a technology for non-diagnostic or therapeutic purposes, comprising the steps of:
(1) the 69/70-crRNA-W and 69/70-crRNA-M sequences of wild type non-mutant 69/70 and mutant 69/70 deletion site specificity are designed aiming at the genome sequences of wild strains and mutant strains of the new coronavirus, and compared with the traditional crRNA design, the PAM sequence beside the sequences is TTC instead of the traditional TTTV. The designed crRNA sequence is shown as SEQ ID NO: 1-2, constructing 69/70-crRNA-W and 69/70-crRNA-M in vitro transcription vectors, and performing in vitro transcription and purification, or directly synthesizing;
(2) designing RT-RAA primers aiming at the non-mutation 69/70 and mutation target 69/70 deletion of the new coronavirus in the step (1), wherein the RT-RAA primers are sequences with low mutation frequency (less than one thousandth) screened out by carrying out high-throughput sequence comparison on the new coronavirus and counting the mutation frequency of each base. And according to the design principle of RT-RAA primers, a sequence with good amplification efficiency and high specificity is obtained by screening and verifying the low-mutation-frequency and specific sequences and is used as the RT-RAA primers, and the primer sequence is shown as SEQ ID NO: 3-4, performing RT-RAA reaction on a nucleic acid sample to be detected to obtain an RT-RAA reaction product; or designing RT-PCR primers aiming at the non-mutated 69/70 and mutated target 69/70 deletion of the new coronavirus in the step (1), wherein the RT-PCR primers are sequences with low mutation frequency (less than one ten thousandth) screened out by carrying out high-throughput sequence alignment on the new coronavirus and counting the mutation frequency of each base. And according to the design principle of RT-PCR primers, a sequence with good amplification efficiency and high specificity is obtained by screening and verifying the low-mutation-frequency and specific sequences and is used as the RT-PCR primers, and the primer sequence is shown as SEQ ID NO: 5-6, performing RT-PCR reaction on a nucleic acid sample to be detected to obtain an RT-PCR reaction product;
(3) mixing the purified crRNA in-vitro transcription product or synthesized 69/70-crRNA-W or 69/70-crRNA-M molecule of the step (1), the RT-RAA or RT-PCR reaction product of the step (2), LbaCas12a and a report single-stranded DNA molecule in a proper system in a proper proportion for reaction; selecting optimal reaction time through continuous kinetic study in the reaction process;
(4) and detecting the reaction product to obtain a detection result.
Preferably, in step (1), the NCBI accession number of the genome of the new coronavirus wild strain is NC _045512.2, and the NCBI accession number of the mutant reference gene sequence is MZ 773928.1.
Preferably, the synthesis method of 69/70-crRNA-W and 69/70-crRNA-M sequences to be designed in the step (1) comprises the following steps: 69/70-crRNA-W and 69/70-crRNA-M in vitro transcription vectors are constructed and transcribed and purified in vitro, or synthesized directly chemically, but not limited thereto.
Preferably, the nucleic acid sample to be detected in step (2) may be nucleic acid extracted from a clinical sample, or a sample treated by a sample treatment method in other nucleic acid detection means.
Preferably, the LbaCas12a described in step (3) can be obtained by recombinant expression and purification or use the LbaCas12a product of NEB;
preferably, the design of the report single-stranded DNA molecule in step (3): when the DNA is used for detecting lateral flow immunochromatographic test paper, the 12-base random sequence single-stranded DNA molecule 5 '-Digoxin-NNNNNNNNNNNN-Biotin-3' (SEQ ID NO: 7) with Digoxin and Biotin groups at two ends respectively is not limited to the above;
when the fluorescent probe is used for fluorescence detection, the single-stranded DNA molecule with 12-base random sequence and two ends respectively provided with FAM and BHQ1 groups is 5 '-FAM-NNNNNNNNNNNN-BHQ 1-3' (SEQ ID NO: 8), but the fluorescent probe is not limited to the above.
Preferably, when the lateral flow immunochromatographic test strip is used for detection, the reaction system in the step (3) is a 40 mu L system, 50-250 nM LbaCas12a, 100-500 nM 69/70-crRNA-W or 69/70-crRNA-M, 2 nM-4 nM report single-stranded DNA molecule, 10U RNase inhibitor (TaKaRa), 5-20 mu L RT-RAA reaction product or RT-PCR reaction product, 1 XNEBuffer 2.1; wherein the molar ratio of LbaCas12a to 69/70-crRNA-W or 69/70-crRNA-M is 1: 2;
further preferably, when the reagent kit is used for lateral flow immunochromatographic test strip detection, the reaction system in the step (3) is a 40-L system, 50nM LbaCas12a, 100nM 69/70-crRNA-W or 69/70-crRNA-M, 2nM reporter single-stranded DNA molecule, 10U RNase inhibitor (TaKaRa), 5. mu.L RT-RAA reaction product or RT-PCR reaction product, 1 XNEBbuffer 2.1;
preferably, when the fluorescent probe is used for fluorescence detection, the reaction system in the step (3) is a 20-microliter system, 50-250 nM LbaCas12a, 100-500 nM 69/70-crRNA-W or 69/70-crRNA-M, 500-1000 nM report single-stranded DNA molecule, 10U RNase inhibitor (TaKaRa), 2.5-10 microliter RT-RAA reaction product or RT-PCR reaction product, and 1 XNEBbuffer 2.1; wherein the molar ratio of LbaCas12a to 69/70-crRNA-W or 69/70-crRNA-M is 1: 2;
further preferably, when used for fluorescence detection, the reaction system described in step (3) is a 20. mu.L system with 50nM LbaCas12a, 100nM 69/70-crRNA-W or 69/70-crRNA-M, 500nM reporter single-stranded DNA molecule, 10U RNase inhibitor (TaKaRa), 2.5. mu.L RT-RAA reaction product or RT-PCR reaction product, 1 XNEBbuffer 2.1;
preferably, the reaction system in the step (3) can be lyophilized, wherein the lyophilized method is that the prepared reaction system (without RT-RAA reaction product or RT-PCR reaction product) is frozen in a refrigerator at-80 ℃ overnight and then vacuum-dried at-50 ℃ for 12 h; when the fluorescent probe is used for fluorescence detection, the application method of the freeze-dried reaction system comprises the steps of dissolving the freeze-dried reaction system by 10-17.5 mu L of RNA-free enzyme water, adding 2.5-10 mu L of RT-RAA reaction product or RT-PCR reaction product, and then carrying out reaction; when the reagent is used for lateral flow immune test strip detection, the application method of the freeze-dried reaction system comprises the steps of dissolving the freeze-dried reaction system by using 20-35 mu L of RNA-free enzyme water, adding 5-20 mu L of RT-RAA reaction product or RT-PCR reaction product, and then carrying out reaction.
Preferably, the reaction in the step (3) is carried out at 37 ℃ for 10-60 minutes; further 10 to 30 minutes.
Preferably, the design of the lateral flow immunochromatographic test strip in step (4): the lateral flow immunochromatographic test strip adopts a commercial lateral flow chromatography detection test strip (Magigen), and the lateral flow immunochromatographic test strip is sequentially provided with a sample loading area, a Gold-NP anti-digoxin antibody area, a streptavidin strip (namely a detection strip) and an anti-antibody strip (namely a quality control strip).
Preferably, the detection method of the lateral flow immunochromatographic test strip in the step (4) is to soak the sample loading area of the test strip into the reaction volume in the step (3), incubate for 5min at room temperature, and read the strength of the detection strip by naked eyes; or other lateral flow test strips are carried out according to a corresponding color development method.
Preferably, the detection device used in the fluorescence detection in step (4) can be a conventional microplate reader or any fluorescence detection device capable of performing fluorescence excitation and detection on FAM fluorescence channels.
More preferably, the detection conditions for the fluorescence detection in step (4) are as follows: fluorescence was excited using excitation light of 492nm wavelength, and the fluorescence intensity was detected at 522nm wavelength.
The mechanism of the invention is as follows:
the LbaCas12a enzyme can target DNA complementary with the LbaCas12a enzyme under the guidance of a characteristic crRNA sequence, and activate cis-reaction activity and trans-cleavage activity of the LbaCas12a enzyme. The trans-cleavage activity of the probe can nonspecifically cleave a single-stranded DNA probe molecule marked by fluorescence or biotin, so that the probe can be detected by fluorescence or lateral flow immune test paper.
According to the invention, through the capability of the LbaCas12a/crRNA system to specifically recognize DNA targets, 69/70-crRNA-W and 69/70-crRNA-M deleted aiming at a non-mutated target 69/70 and a mutated target 69/70 of the new coronavirus are respectively designed, and when two crRNAs are used for respectively detecting the same sample, whether the nucleic acid sample contains the wild type or the 69/70 deletion mutant strain of the new coronavirus can be effectively distinguished according to the difference of detection results.
Compared with the prior art, the invention has the following advantages and effects:
compared with the existing sequencing method for detecting the deletion mutation of the new coronavirus 69/70, the method provided by the invention has the advantages that the LbaCas12a/crRNA is used for detecting sample molecules, only one PCR instrument or a constant temperature device and a simple fluorescence reading device are needed, the 69/70 deletion mutant strain can be rapidly distinguished from the new coronavirus positive sample, and meanwhile, the method has the characteristics of high specificity, high sensitivity and low cost, and is more widely applicable to various molecular diagnosis laboratories. The invention can quickly and effectively distinguish the wild type and the mutant strain of the new coronavirus, and has important significance for controlling the epidemic situation of the new coronavirus.
Drawings
FIG. 1 shows the specificity of 69/70-crRNA-W and 69/70-crRNA-M targets designed by the novel coronavirus against 69/70 wild-type (WT) and 69/70 deletion Mutant (MT) and their PAM (TTC) sequences; the electrophorogram is used for verifying the cutting effectiveness and specificity of crRNA in vitro, and a 69/70-crRNA-W and 69/70-crRNA-M detection system or an NC control group detection system without adding crRNA is added into 69/70 wild type or mutant substrates.
FIG. 2 shows the results of fluorescence detection of the wild-type and 69/70-deleted mutants of the novel coronavirus 69/70 by RT-PCR-Cas12a nucleic acid detection system in example 1; wherein, the detection samples are 69/70 deletion targets (MT) with different concentrations, and the RT-RCR amplification primers adopt the nucleotide sequences shown in SEQ ID NO: 5-6; n-3, concentration of target nucleic acid 10-14M，10-15M，10-16M，10-17M，10-18M, BC (blank control), error bars: ± SD,. x: p is a radical of<0.0001。
FIG. 3 shows the results of fluorescence detection of the wild-type and 69/70-deleted mutants of the novel coronavirus 69/70 in RT-RAA-Cas12a nucleic acid detection system in example 1; wherein the test samples are of different concentrations69/70 deletion of target (MT), RT-RAA amplification primers used SEQ ID NO: 3-4; n-3, concentration of target nucleic acid 10-14M，10-15M，10-16M，10-17M, BC (blank control), error bars: ± SD,. x: p is a radical of<0.0001。
FIG. 4 shows the results of detection of the mutant strain of neocoronavirus 69/70 wild type and 69/70 deletion by lateral flow dipstick method in RT-PCR-Cas12a nucleic acid detection system in example 2; wherein, the detection samples are 69/70 (MT) with different concentrations, and the RT-PCR amplification primers adopt the nucleotide sequences shown in SEQ ID NO: 5-6; target nucleic acid concentration of 10-14M，10-15M，10-16M，10-17M, BC (blank control); w and M correspond to 69/70-crRNA-W and 69/70-crRNA-M, respectively.
FIG. 5 shows the results of lateral flow dipstick assay of RT-RAA-Cas12a nucleic acid detection system in example 2 for detecting mutant strains of neocoronavirus 69/70 wild type and 69/70 deletion; wherein, the detection samples are 69/70 deletion targets (MT) with different concentrations, and the RT-RAA amplification primers adopt the nucleotide sequences shown in SEQ ID NO: 3-4; target nucleic acid concentration of 10-15M，10-16M; w and M correspond to 69/70-crRNA-W and 69/70-crRNA-M, respectively.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
Example 1:
in the embodiment of the invention, the CRISPR/Cas12a technology-based method for rapidly detecting the characteristic sequence nucleic acid of the wild type and the mutant strain of the novel coronavirus, is used for non-diagnosis or treatment and comprises the following steps:
(1) characteristic 69/70-crRNA-W and 69/70-crRNA-M were designed against the wild type (codon ACATGT of 69/70) and 69/70 deletion mutation of the new coronavirus (SARS-CoV-2)69/70, respectively, and unlike the conventional crRNA design, the PAM sequence next to this sequence was TTC instead of the conventional TTTV, as shown in fig. 1 and SEQ ID NO: 1-2, constructing 69/70-crRNA-W and 69/70-crRNA-M transcription vectors, and then transcribing and purifying.
TABLE 1 crRNA sequence, RT-RAA primer, RT-PCR primer sequence
Specifically, the method for crRNA in vitro transcription comprises the following steps: the reaction was carried out for 16 hours at 37 ℃ in the following system: NTPs were each 1.5. mu.L, 10 × reaction buffer 1.5. mu.L, Template DNA 1. mu.g, T7 RNA Polymerase Mix 1.5. mu.L, and nucleic-free water added to 20. mu.L.
Specifically, the purification method of the crRNA transcript comprises the following steps: after the transcription product was treated with DNaseI (TaKaRa) for 15 minutes, the transcribed crRNA was purified using NEB RNA clean Kit or other RNA purification Kit.
The cleavage effectiveness of the designed crRNA target is verified, and 50nM LbaCas12a, 100nM 69/70-crRNA-W or 69/70-crRNA-M, 5nM 69/70 wild type (69/70-WT) or 69/70 mutant (69/70-MT) linearized DNA double strand, 10U RNase inhibitor (TaKaRa), 1 XNEBbuffer 2.1 are adopted in a 20 mu L system. The reaction conditions were 37 ℃ for 60 min. After the reaction is finished, taking a proper amount of reaction system for gel electrophoresis. The results are shown in FIG. 1, which indicates that designed 69/70-crRNA-W or 69/70-crRNA-M can effectively distinguish between wild-type 69/70 and 69/70 deletion mutant DNA targets.
Wherein the linearized DNA duplex is a DNA molecule comprising a designed targeting segment.
(2) Aiming at published new coronavirus genomes (genome sequence information is derived from new coronavirus sequences submitted by GISAID countries (as far as 2021, 01, 13 days), 360482 sequences are totally contained, https:// www.gisaid.org /) sequences, relatively conservative and specific regions are selected near a 69/70-crRNA-W/M target point to serve as primers of RT-RAA, the RT-RAA primers are sequences with low mutation frequency (less than one thousandth) screened out through new coronavirus high-throughput sequence comparison and statistics of mutation frequency of each base. And then according to the design principle of RT-RAA primers, sequences with good amplification efficiency and high specificity are obtained by screening and verifying from the low mutation frequency and specific sequences and are used as RT-RAA primers, and SEQ ID NO: 3 to 4.
The pretreatment method of the nucleic acid sample to be detected is to carry out RT-RAA amplification on 2 mu L of sample solution, and the obtained amplification product is the processed nucleic acid sample; the nucleic acid sample to be tested is in this example an in vitro transcribed RNA molecule comprising a designed targeting fragment.
RT-RAA reaction system: in a 50. mu.L system, 400nM of the upstream primer (69/70-RT-RAA-F), 400nM of the downstream primer (69/70-RT-RAA-R), 41.5. mu.L of A Buffer (purchased from Mass-testing Bio Inc.) and 2. mu.L of the template of the nucleic acid sample to be tested were used. The above systems were mixed and added to lyophilized RT-RAA reaction microspheres, magnesium acetate MgAc 280mM 2.5. mu.L was added and the reaction was started at 37 ℃.
Or, aiming at the published genome sequence of the new coronavirus, selecting a relatively conservative and specific region near the 69/70-crRNA-W/M target point as a primer of RT-PCR, wherein the RT-PCR primer is a sequence which is obtained by counting the mutation frequency of each base through high-throughput sequence alignment of the new coronavirus, and the screened mutation frequency is low (less than one ten thousandth). And then according to the design principle of RT-PCR primers, sequences with good amplification efficiency and high specificity are obtained by screening and verifying from the specific sequences with low mutation frequency and are used as RT-PCR primers, and SEQ ID NO: 5 to 6.
The pretreatment method of the nucleic acid sample to be detected is that 18.5 mu L of sample solution is subjected to RT-PCR amplification, and the obtained amplification product is the processed nucleic acid sample; the nucleic acid sample to be tested is in this example an in vitro transcribed RNA molecule comprising a designed targeting fragment.
RT-PCR reaction system: 50 μ L of the system, 400nM of the forward primer (69/70-RT-PCR-F), 400nM of the reverse primer (69/70-RT-PCR-R), 25 μ L of 2 XBuffer mix (containing Buffer system and dNTP), 2.5 μ L of enzyme mix (containing reverse transcriptase, RNase inhibitor and DNA polymerase), and 18.5 μ L of the nucleic acid template to be tested.
RT-PCR reaction procedure: 30min at 50 ℃; 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 30 s; 5min at 72 ℃; storing at 4 ℃.
(3) Design of reporter single stranded DNA molecules: 12-base random single-stranded DNA molecule 5 '-FAM-NNNNNNNNNNNN-BHQ 1-3' (SEQ ID NO: 8) with FAM and BHQ1 groups at both ends respectively.
(4) The 69/70-crRNA-W or 69/70-crRNA-M in-vitro transcription product, the treated nucleic acid sample, the LbaCas12a and the reporting single-stranded DNA molecule are mixed in a proper system in a proper proportion for reaction.
(5) The reaction system is as follows: 50nM LbaCas12a, 100nM 69/70-crRNA-W or 69/70-crRNA-M, 500nM reporter single stranded DNA molecule, 10U RNase inhibitor (TaKaRa), 2.5. mu.L treated nucleic acid sample, 1 XNEBbuffer 2.1 in 20. mu.L system. The reaction system is reacted at 37 ℃ for 10 to 15 minutes.
(6) The reaction product was subjected to fluorescence excitation using excitation light of a wavelength of 492nm in a fluorescence detection device, and the fluorescence intensity was detected at a wavelength of 522nm to obtain a detection result.
(7) Taking RT-PCR amplification primers (SEQ ID NO: 5-6) as an example, the reaction time of the step (5) is 10min, and the result is shown in FIG. 2, which shows that the concentration of the target nucleic acid is 10-17M and above, the detection method can effectively distinguish wild 69/70 and 69/70 deletion mutant new coronavirus.
(8) Taking RT-RAA amplification primers (SEQ ID NO: 3-4) as an example, the reaction time in the step (5) is 15min, and the result is shown in FIG. 3, which shows that the concentration of the target nucleic acid is 10-17M and above can effectively distinguish 69/70 wild type and 69/70 deletion mutant type new coronavirus through the detection method.
Example 2:
in the embodiment of the invention, the CRISPR/Cas12a technology-based method for rapidly detecting the characteristic sequence nucleic acid of the wild type and the mutant strain of the novel coronavirus, is used for non-diagnosis or treatment and comprises the following steps:
(1) characteristic 69/70-crRNA-W and 69/70-crRNA-M were designed against the wild type (codon ACATGT of 69/70) and 69/70 deletion mutation of the new coronavirus (SARS-CoV-2)69/70, respectively, and unlike the conventional crRNA design, the PAM sequence next to this sequence was TTC instead of the conventional TTTV, as shown in fig. 1 and SEQ ID NO: 1-2, constructing 69/70-crRNA-W and 69/70-crRNA-M transcription vectors, and then transcribing and purifying.
Specifically, the method for crRNA in vitro transcription comprises the following steps: the reaction was carried out for 16 hours at 37 ℃ in the following system: nuclean-free water was added to 20. mu.L each of 1.5. mu.L of NTP, 1.5. mu.L of 10 × reaction buffer, 1. mu.g of Template DNA, and 1.5. mu.L of T7 RNA Polymerase Mix.
Specifically, the purification method of the crRNA transcript comprises the following steps: after the transcription product was treated with DNaseI (TaKaRa) for 15 minutes, the transcribed crRNA was purified using NEB RNA clean Kit or other RNA purification Kit.
(2) Aiming at the published genome sequence of the new coronavirus, selecting a relatively conservative and specific region near a target point 69/70-crRNA-W/M as a primer of RT-RAA, wherein the RT-RAA primer is a sequence which is obtained by counting the mutation frequency of each base through high-throughput sequence comparison of the new coronavirus, and the screened mutation frequency is low (less than one thousandth). And then according to the design principle of RT-RAA primers, sequences with good amplification efficiency and high specificity are obtained by screening and verifying from the low mutation frequency and specific sequences and are used as RT-RAA primers, and SEQ ID NO: 3 to 4.
The pretreatment method of the nucleic acid sample to be detected is to carry out RT-RAA amplification on 2 mu L of sample solution, and the obtained amplification product is the processed nucleic acid sample; the nucleic acid sample to be tested is in this example an in vitro transcribed RNA molecule comprising a designed targeting fragment.
RT-RAA reaction system: in a 50-mu L system, 400nM of the upstream primer (69/70-RT-RAA-F), 400nM of the downstream primer (69/70-RT-RAA-R), 41.5 mu L of A Buffer and 2 mu L of the nucleic acid sample template to be detected are included. The above systems were mixed and added to lyophilized RT-RAA reaction microspheres, magnesium acetate MgAc 280mM 2.5. mu.L was added and the reaction was started at 37 ℃.
Or, aiming at the published genome sequence of the new coronavirus, selecting a relatively conservative and specific region near the 69/70-crRNA-W/M target point as a primer of RT-PCR, wherein the RT-PCR primer is a sequence which is obtained by counting the mutation frequency of each base through high-throughput sequence alignment of the new coronavirus, and the screened mutation frequency is low (less than one ten thousandth). And then according to the design principle of RT-PCR primers, sequences with good amplification efficiency and high specificity are obtained by screening and verifying from the specific sequences with low mutation frequency and are used as RT-PCR primers, and SEQ ID NO: 5 to 6.
The pretreatment method of the nucleic acid sample to be detected is that 18.5 mu L of sample solution is subjected to RT-PCR amplification, and the obtained amplification product is the processed nucleic acid sample; the nucleic acid sample to be tested is in this example an in vitro transcribed RNA molecule comprising a designed targeting fragment.
RT-PCR reaction system: 50 μ L of the system, 400nM of the forward primer (69/70-RT-PCR-F), 400nM of the reverse primer (69/70-RT-PCR-R), 25 μ L of 2 XBuffer mix (containing Buffer system and dNTP), 2.5 μ L of enzyme mix (containing reverse transcriptase, RNase inhibitor and DNA polymerase), and 18.5 μ L of the nucleic acid template to be tested.
RT-PCR reaction procedure: 30min at 50 ℃; 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 30 s; 5min at 72 ℃; storing at 4 ℃.
(3) Design of reporter single stranded DNA molecules: 12-base random single-stranded DNA molecule 5 '-Digoxin-NNNNNNNNNNNN-Biotin-3' (SEQ ID NO: 7) with Digoxin and Biotin groups at both ends, respectively.
(4) The 69/70-crRNA-W or 69/70-crRNA-M in-vitro transcription product, the treated nucleic acid sample, the LbaCas12a and the reporting single-stranded DNA molecule are mixed in a proper system in a proper proportion for reaction.
(5) The reaction system is as follows: 50nM LbaCas12a, 100nM 69/70-crRNA-W or 69/70-crRNA-M, 2nM reporter single stranded DNA molecule, 10U RNase inhibitor (TaKaRa), 5. mu.L treated nucleic acid sample, 1 XNEBbuffer 2.1 in 40. mu.L system. The reaction system was reacted at 37 ℃ for 30 minutes.
(6) And soaking the lateral flow immunochromatography test paper sample loading area into the reaction mixed liquid, incubating for 5min at room temperature, and reading the test paper strip by naked eyes to obtain a detection result.
(7) Taking RT-PCR amplification primers (SEQ ID NO: 5-6) as an example, the results are shown in FIG. 4, which shows that the concentration of the target nucleic acid is 10-16M and aboveThe detection method can effectively distinguish 69/70 wild type and 69/70 deletion mutant type new coronavirus.
(8) Taking RT-RAA amplification primers (SEQ ID NOS: 3-4) as an example, the results are shown in FIG. 5, which indicates that the concentration of the target nucleic acid is 10-16M and above can effectively distinguish 69/70 wild type and 69/70 deletion mutant type new coronavirus through the detection method.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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Claims (10)
1. A kit for rapidly detecting wild type and mutant strain of a novel coronavirus based on CRISPR/Cas12a technology is characterized in that: comprises LbaCas12a, a 69/70-crRNA-W sequence specific to 69/70 locus wild type, a 69/70-crRNA-M sequence specific to 69/70 deletion mutant strain, RT-RAA primers or RT-PCR primers specific to new coronavirus wild type and mutant strain characteristic sequences, and a report single-stranded DNA molecule;
the specific 69/70-crRNA-W sequence is shown as SEQ ID NO: 1, the sequence of the specificity 69/70-crRNA-M is shown as SEQ ID NO: 2 is shown in the specification;
the RT-RAA primer aiming at the wild type and mutant strain characteristic sequences of the new coronavirus is SEQ ID NO: 69/70-RT-RAA-F/R primer groups shown in 3-4;
the RT-PCR primer aiming at the new coronavirus wild type and mutant strain characteristic sequences is SEQ ID NO: 5-6, wherein the primer group is 69/70-RT-PCR-F/R;
the 69/70-RT-RAA-F/R primer group is used for amplifying a nucleic acid fragment containing complementarity with 69/70-crRNA-W and/or 69/70-crRNA-M;
the 69/70-RT-PCR-F/R primer group is used for amplifying a nucleic acid fragment containing complementarity with 69/70-crRNA-W and/or 69/70-crRNA-M.
2. The kit for rapid detection of wild type and mutant strains of novel coronavirus based on CRISPR/Cas12a technology as claimed in claim 1, characterized in that:
the report single-stranded DNA molecule is SEQ ID NO: 7 or SEQ ID NO: 8.
3. a method for rapidly detecting a new coronavirus wild type and a new coronavirus mutant based on CRISPR/Cas12a technology, which is characterized by comprising the following steps: the method is used for non-diagnostic or therapeutic purposes and comprises the following steps:
(1) aiming at genome sequences of wild strains and mutant strains of the new coronavirus, 69/70-crRNA-W and 69/70-crRNA-M sequences with deletion site specificity of wild type non-mutant 69/70 and mutant strain 69/70 are designed, wherein the designed crRNA sequences are shown as SEQ ID NO: 1-2, constructing 69/70-crRNA-W and 69/70-crRNA-M in vitro transcription vectors, and performing in vitro transcription and purification, or directly synthesizing;
(2) designing an RT-RAA primer aiming at the deletion of the non-mutant 69/70 and mutant target 69/70 of the novel coronavirus in the step (1), wherein the primer sequence is shown as SEQ ID NO: 3-4, performing RT-RAA reaction on a nucleic acid sample to be detected to obtain an RT-RAA reaction product; or designing RT-PCR primers aiming at the non-mutation 69/70 and mutation target 69/70 deletion of the novel coronavirus in the step (1), wherein the primer sequences are shown as SEQ ID NO: 5-6, performing RT-PCR reaction on a nucleic acid sample to be detected to obtain an RT-PCR reaction product;
(3) mixing the purified crRNA in-vitro transcription product or synthesized 69/70-crRNA-W or 69/70-crRNA-M molecule of the step (1), the RT-RAA or RT-PCR reaction product of the step (2), LbaCas12a and a report single-stranded DNA molecule in a proper system in a proper proportion for reaction;
(4) the reaction product is detected by fluorescence or lateral flow immunochromatographic test paper to obtain a detection result.
4. The method for rapid detection of new coronavirus wild type and mutant strain based on CRISPR/Cas12a technology as claimed in claim 3, characterized in that:
designing a report single-stranded DNA molecule in the step (3): when the DNA is used for lateral flow immunochromatographic test paper detection, the 12-base random sequence single-stranded DNA molecule 5 '-Digoxin-NNNNNNNNNNNN-Biotin-3' with Digoxin and Biotin groups at two ends is shown in SEQ ID NO: 7;
when the fluorescent probe is used for fluorescent detection, the two ends of the single-stranded DNA molecule with the 12-base random sequence and the FAM and BHQ1 groups at the two ends respectively have 5 '-FAM-NNNNNNNNNNNN-BHQ 1-3', and the single-stranded DNA molecule is shown in SEQ ID NO: 8.
5. the method for rapid detection of new coronavirus wild type and mutant strain based on CRISPR/Cas12a technology according to claim 3 or 4, characterized in that:
when the kit is used for lateral flow immunochromatographic test paper detection, the reaction system in the step (3) is a 40 mu L system, 50-250 nM LbaCas12a, 100-500 nM 69/70-crRNA-W or 69/70-crRNA-M, 2 nM-4 nM report single-stranded DNA molecule, 10U RNase inhibitor, 5-20 mu L RT-RAA reaction product or RT-PCR reaction product, and 1 XNEBuffer 2.1; wherein the molar ratio of LbaCas12a to 69/70-crRNA-W or 69/70-crRNA-M is 1: 2;
when the fluorescent probe is used for fluorescence detection, the reaction system in the step (3) is a 20 mu L system, 50-250 nM LbaCas12a, 100-500 nM 69/70-crRNA-W or 69/70-crRNA-M, 500-1000 nM report single-stranded DNA molecule, 10U RNase inhibitor, 2.5-10 mu L RT-RAA reaction product or RT-PCR reaction product, and 1 XNEBbuffer 2.1; wherein the molar ratio of LbaCas12a to 69/70-crRNA-W or 69/70-crRNA-M is 1: 2.
6. the method for rapid detection of new coronavirus wild type and mutant strain based on CRISPR/Cas12a technology as claimed in claim 5, characterized in that:
when the kit is used for lateral flow immunochromatographic test strip detection, the reaction system in the step (3) is a 40-microliter system, 50nM LbaCas12a, 100nM 69/70-crRNA-W or 69/70-crRNA-M, 2nM report single-stranded DNA molecule, 10U RNase inhibitor, 5 microliter RT-RAA reaction product or RT-PCR reaction product, and 1 XNEBbuffer 2.1;
when the fluorescent probe is used for fluorescence detection, the reaction system in the step (3) is a 20-mu-L system, 50nM LbaCas12a, 100nM 69/70-crRNA-W or 69/70-crRNA-M, 500nM reporter single-stranded DNA molecule, 10U RNase inhibitor, 2.5 mu L RT-RAA reaction product or RT-PCR reaction product, and 1 XNEBbuffer 2.1.
7. The method for rapid detection of new coronavirus wild type and mutant strain based on CRISPR/Cas12a technology according to claim 3 or 4, characterized in that:
freeze-drying the reaction system in the step (3), wherein the freeze-drying method is that the prepared reaction system without the RT-RAA reaction product or the RT-PCR reaction product is placed in a refrigerator at the temperature of-80 ℃ for freezing overnight and then is dried in vacuum at the temperature of-50 ℃ for 12 hours; when the fluorescent probe is used for fluorescence detection, the application method of the freeze-dried reaction system comprises the steps of dissolving the freeze-dried reaction system by 10-17.5 mu L of RNA-free enzyme water, adding 2.5-10 mu L of RT-RAA reaction product or RT-PCR reaction product, and then carrying out reaction; when the reagent is used for lateral flow immune test strip detection, the application method of the freeze-dried reaction system comprises the steps of dissolving the freeze-dried reaction system by using 20-35 mu L of RNA-free enzyme water, adding 5-20 mu L of RT-RAA reaction product or RT-PCR reaction product, and then carrying out reaction.
8. The method for rapid detection of new coronavirus wild type and mutant strain based on CRISPR/Cas12a technology according to claim 3 or 4, characterized in that:
the reaction condition in the step (3) is that the reaction is carried out for 10-60 minutes at 37 ℃.
9. The method for rapid detection of new coronavirus wild type and mutant strain based on CRISPR/Cas12a technology according to claim 3 or 4, characterized in that:
the detection method of the lateral flow immunochromatographic test paper in the step (4) comprises the steps of soaking a test paper sample loading area into the reaction volume in the step (3), incubating at room temperature for 5min, and reading the strength of a detection strip by naked eyes; or other lateral flow test paper is carried out according to a corresponding color development method;
and (4) detecting the fluorescence in the step (4) by using a microplate reader or any fluorescence detection device capable of performing fluorescence excitation and detection on the FAM fluorescence channel.
10. The method for rapid detection of new coronavirus wild-type and mutant strains based on CRISPR/Cas12a technology according to claim 9, characterized in that:
the detection conditions adopted by the fluorescence detection in the step (4) are as follows: fluorescence was excited using excitation light of 492nm wavelength, and the fluorescence intensity was detected at 522nm wavelength.
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