WO2023207909A1 - Crispr-based nucleic acid detection kit and use thereof - Google Patents

Crispr-based nucleic acid detection kit and use thereof Download PDF

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WO2023207909A1
WO2023207909A1 PCT/CN2023/090320 CN2023090320W WO2023207909A1 WO 2023207909 A1 WO2023207909 A1 WO 2023207909A1 CN 2023090320 W CN2023090320 W CN 2023090320W WO 2023207909 A1 WO2023207909 A1 WO 2023207909A1
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nucleic acid
reaction
amplification
lysis
detection
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PCT/CN2023/090320
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French (fr)
Chinese (zh)
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刘佳
瞿介明
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上海科技大学
上海交通大学医学院附属瑞金医院
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
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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
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to an extraction-free and cap-opening CRISPR-based nucleic acid detection kit and its application.
  • SARS-CoV-2 The pathogen causing COVID-19 has been confirmed to be a beta coronavirus, named SARS-CoV-2. Similar to other coronaviruses, SARS-CoV-2 is a single-stranded, positive-sense RNA virus. SARS-CoV-2 and SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) share 79% and 50% nucleic acid similarity, respectively.
  • RT-PCR reverse transcription PCR
  • the E, N and ORF1ab genes of SARS-CoV-2 are commonly used RT-PCR target genes. High-throughput RT-PCR platforms have also been developed for large-scale diagnosis.
  • RT-PCR usually relies on special equipment, so large-scale RT-PCR testing is often limited to patients in hospitals.
  • the current epidemic prevention and control requirements are nucleic acid test results within 48 hours, and the lack of testing methods for high-risk environments (such as airports, high-speed rail and other public places) also brings great uncertainty to epidemic prevention and control.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR-associated genes
  • the modular nature of CRISPR makes this technology widely used in genome engineering.
  • CRISPR-based molecular diagnosis of pathogenic nucleic acids relies on the RNA or DNA targeting activity of Cas nuclease, which can achieve rapid and accurate detection of the new coronavirus within 30 minutes.
  • CRISPR has not yet been widely used.
  • One of the main limitations is that the operation of CRISPR is too complex.
  • the technical problem to be solved by the present invention is to overcome the lack of an easy-to-operate, accurate and fast CRISPR reaction system in the existing technology, and provide a CRISPR-based nucleic acid detection kit and its application.
  • the nucleic acid detection kit of the present invention can quickly and accurately reflect the detection results of CRISPR. For example, when detecting SARS-CoV-2, specific crRNA is used to identify viral genes with high sensitivity and specificity; and it can eliminate the need for nucleic acid extraction and opening of the lid. Detection reduces the possibility of contamination and false positive rate, and has high application value.
  • the present invention solves the above technical problems through the following technical solutions.
  • a first aspect of the present invention provides a gene editing system, which includes a nuclease and a guide RNA; wherein, the sequence of the guide RNA is selected from the nucleotide sequences shown in SEQ ID NO: 1 to 4 of one or more.
  • the sequence of the guide RNA is as shown in any one of SEQ ID NO: 1 to 4.
  • the nuclease may be an enzyme conventionally used in the art to destroy phosphodiester bonds in nucleic acids to cause cleavage of nucleic acid chains, and is preferably Cas protein.
  • the Cas protein is Cas12a or Cas13a;
  • the Cas12a is preferably AsCas12a (from Acidaminococcus sp.BV3L6), BbCas12a (from Beauveria bassiana KA00251), BoCas12a (from Bacteroidetes oral), FnCas12a (from Francisella novicida U112), HkCas12a (from Helcococcus kunzii), Lb4Cas12a (from Lachnospiraceae bacterium MC2017), Lb5Cas12a (from Lachnospiraceae bacterium NC2008), LbCas12a (from Lachnospiraceae bacterium ND2006), OsCas12a (from Oribacterium sp.) or TsCas12a (from Thiomicrospira sp.XS5).
  • AsCas12a from Acidaminococcus sp.BV3L6
  • BbCas12a from Beauveri
  • a second aspect of the invention provides an isolated nucleic acid encoding the gene editing system of the first aspect.
  • a third aspect of the present invention provides a detection system for nucleic acid detection.
  • the detection system includes a gene editing system and a reaction buffer; wherein the gene editing system includes a nuclease and a guide RNA, and the reaction buffer includes 10 ⁇ 50mM NaCl, 5 ⁇ 50mM MgCl 2 , 5 ⁇ 50mM Tris-HCl, 0.1 ⁇ 1.0mM dithiothreitol and 50 ⁇ 200 ⁇ g/mL bovine serum albumin, pH 7 ⁇ 8.
  • the nuclease is as described in the first aspect
  • the guide RNA is a conventional RNA in the art that can bind the nuclease and guide the nuclease to the target gene, such as sgRNA or crRNA;
  • the gene of interest may be a viral gene, such as a gene of an RNA virus.
  • the gene editing system is the gene editing system described in the first aspect.
  • the working concentration of the guide RNA and the nuclease can be conventional in the art, for example, the working concentration of the guide RNA is 50-200 nM, such as 100 nM; the working concentration of the nuclease is 25-100 nM, For example 50nM.
  • the reaction buffer includes 20-30mM NaCl, 15-25mM MgCl 2 , 10-20mM Tris-HCl, 0.5-0.75mM dithiothreitol and 100-200 ⁇ g/mL Bovine serum albumin, pH 7-8.
  • the reaction buffer includes 20mM NaCl, 15mM MgCl 2 , 10mM Tris-HCl, 0.5mM dithiothreitol and 100 ⁇ g/mL bovine serum albumin, pH 7.9.
  • the fourth aspect of the present invention provides an amplification system for nucleic acid amplification.
  • the amplification system includes primers, RNase H and RPA enzyme; wherein, the primers are used to amplify nucleic acids in the sample to be tested, so
  • the RPA enzyme is an enzyme used for recombinase polymerase amplification, and the concentration of RNase H is not higher than 1U/ ⁇ L.
  • the RPA enzyme can be an enzyme conventionally used in the field for recombinase polymerase amplification (RPA), including recombinase capable of binding single-stranded nucleic acid primers, single-stranded DNA binding protein (ssDNA binding) protein, SSB) and strand-displacement DNA polymerase.
  • RPA recombinase polymerase amplification
  • the concentration of RNase H is 0.1 ⁇ 0.5U/ ⁇ L.
  • the primer is a primer used to amplify the nucleic acid of SARS-CoV-2; preferably, it is a primer used to amplify the N gene of SARS-CoV-2.
  • the primers include a forward primer and a reverse primer; the forward primer is preferably selected from one or more of the nucleotide sequences shown in SEQ ID NO: 5 to 8, and the reverse primer is preferably selected from the group consisting of One or more of the nucleotide sequences shown in SEQ ID NO:9 ⁇ 12.
  • the nucleotide sequence of the forward primer is as shown in SEQ ID NO:5, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO:9; or, the The nucleotide sequence of the forward primer is shown in SEQ ID NO: 6, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 10; or, the nucleotide sequence of the forward primer is shown in SEQ ID NO:7, the nucleotide sequence of the reverse primer is shown in SEQ ID NO:11; or, the nucleotide sequence of the forward primer is shown in SEQ ID NO:8, the reverse primer The nucleotide sequence of the primer is shown in SEQ ID NO:12.
  • the working concentration of the primer can be conventional in the art, preferably 0.2-2 ⁇ M, such as 0.4 ⁇ M.
  • the amplification system further includes a reverse transcriptase; when the nucleic acid to be tested is RNA, the reverse transcriptase is used to reverse transcribe RNA into DNA.
  • the fifth aspect of the present invention provides a lysis system for pathogen lysis.
  • the lysis system includes a pathogen transfer solution and a lysis solution; the pathogen transfer solution is used to maintain the pathogenic activity of the sample to be tested, and the lysis solution is used to The pathogenic nucleic acid in the sample to be tested is exposed; wherein, the volume ratio of the pathogen transfer solution and the lysis solution is 1: (0.5-10).
  • the pathogen transfer solution and lysis solution are conventional in this field.
  • the pathogen transfer solution is the pathogen transfer solution of Beijing Youkang, MT0301
  • the lysis solution is the lysis solution of Epicentre, QE09050.
  • the volume ratio of the pathogen transfer solution and the lysis solution is 1: (1-5); preferably, it is 1:1.
  • the pathogen can be a conventional pathogenic organism in the field, preferably selected from pathogenic fungi, viruses and pathogenic prokaryotes; the pathogenic prokaryotes preferably include bacteria, mycoplasma and chlamydia;
  • the pathogen is a virus, preferably an RNA virus, such as SARS-CoV-2, IAV (Influenza A virus) or IBV (Influenza B virus).
  • RNA virus such as SARS-CoV-2, IAV (Influenza A virus) or IBV (Influenza B virus).
  • a sixth aspect of the present invention provides a kit for nucleic acid detection, which kit includes the detection system as described in the third aspect.
  • the gene editing system in the detection system is as described in the first aspect.
  • the detection system further includes a nucleic acid probe.
  • the nucleic acid probe is a probe with a nucleic acid sequence as a backbone conventionally used in this field, and is preferably a fluorescently labeled single-stranded DNA (ssDNA).
  • the fluorescent label refers to connecting a luminescent group and a quenching group to both ends of the single-stranded DNA respectively.
  • the luminescent group can be FAM
  • the quenching group can be BHQ1.
  • the kit further includes an amplification system, which includes an amplification primer and an RPA enzyme; wherein the amplification primer is used to amplify the nucleic acid in the sample to be tested, and the amplification system RPA enzyme is an enzyme used for recombinase polymerase amplification; the amplification system is preferably as described in the fourth aspect.
  • the kit further includes a lysis system, which includes a pathogen transfer solution and a lysis solution; wherein the pathogen transfer solution is used to maintain the pathogenic activity of the sample to be tested, and the lysis solution For exposing the pathogenic nucleic acid in the sample to be tested; the lysis system is preferably as described in the fifth aspect.
  • the kit includes a detection system, an amplification system and a lysis system, and the detection system, amplification system and lysis system are as described above.
  • the seventh aspect of the present invention provides a crRNA for detecting SARS-CoV-2, the crRNA is a crRNA for detecting the N gene of SARS-CoV-2; wherein, the crRNA is selected from the group consisting of SEQ ID NO: 1 to 4 One or more of the nucleotide sequences shown.
  • the crRNA is a nucleotide sequence as shown in any one of SEQ ID NO: 1 to 4.
  • An eighth aspect of the present invention provides a nucleic acid detection method, which method includes: reacting a sample to be tested with the detection system as described in the third aspect, and collecting the fluorescence signal generated by the reaction.
  • the reaction time is 5 to 25 minutes; for example, 10 to 25 minutes, 10 to 20 minutes, or 15 to 20 minutes.
  • the reaction temperature is 35-45°C; for example, 35-42°C, 37-42°C or 39-42°C.
  • the method further includes amplifying the nucleic acid in the sample to be tested in the amplification system as described in the fourth aspect before performing the reaction.
  • the method further includes, before performing the reaction, the sample to be tested is lysed using the cleavage system as described in the fifth aspect to expose the nucleic acid in the sample to be tested, so as to achieve Nucleic acid extraction-free testing.
  • the method further includes, before performing the reaction, the nucleic acid in the sample to be tested is amplified in the amplification system as described in the fourth aspect; and before the amplification reaction, The sample to be tested is lysed with the cleavage system as described in the fifth aspect to expose the nucleic acid in the sample to be tested.
  • the reaction volume ratio of the amplification system and the detection system is 1:(1-5); preferably 1:(2-4), for example, 1:3.
  • the amplification system is pre-stored in a closed container where the detection reaction occurs.
  • the detection system is added into the closed container after the amplification is completed, preferably by injection, to achieve detection without opening the lid.
  • the pyrolysis temperature is 37°C to 95°C, for example, 65°C to 95°C.
  • a ninth aspect of the present invention provides a gene editing system as described in the first aspect, a nucleic acid as described in the second aspect, a detection system as described in the third aspect, an amplification system as described in the fourth aspect, Application of the lysis system as described in the fifth aspect, the kit as described in the sixth aspect, or the crRNA as described in the seventh aspect in preparing reagents for detecting pathogenic nucleic acids.
  • the pathogen is as described in the fifth aspect.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the kit of the present invention uses crRNA targeting the N gene of SARS-CoV-2 and can recognize and cleave this gene in RNA viruses with high sensitivity and specificity (in the preferred embodiment, the LOD is reduced from 100 copies/ ⁇ L (1copy/ ⁇ L), and can detect wild-type and mutant strains of SARS-CoV-2 in a broad spectrum;
  • the optimized reaction system of the present invention can improve detection efficiency while reducing detection limit (up to 1 copy/ ⁇ L) within shortened reaction time and reduced reaction temperature, and has a low false positive rate;
  • the sensitivity can reach 95.92%, the highest positive coincidence rate can reach 95.56%, and the highest negative coincidence rate can reach 100%, with excellent detection efficiency.
  • the kit of the present invention can realize continuous, airtight (no need to open the lid) closed-tube detection, achieve compatibility of various reactions by optimizing the reaction system, and can realize nucleic acid detection without nucleic acid extraction, which has high application value. .
  • Figure 1 is a schematic diagram of the crRNA sequence optimization results of the present invention for the N gene of SARS-CoV-2.
  • Figure 2 is a schematic diagram of the results of the specific detection of SARS-CoV-2 using optimized crRNA and primers for the N gene of SARS-CoV-2 according to the present invention.
  • Figure 3 is a schematic diagram of the results of broad-spectrum detection of SARS-CoV-2 wild type and various mutant strains using optimized crRNA and primers for the N gene of SARS-CoV-2 according to the present invention.
  • Figure 4A- Figure 4I are optimization schematic diagrams of the method of the present invention.
  • Figure 4A is a schematic diagram of the reaction system and process design that requires no extraction and no need to open the lid;
  • Figure 4B is a schematic diagram of the amplification time optimization results
  • Figure 4C is a schematic diagram of the detection reaction time optimization results
  • Figure 4D is a schematic diagram of the results of RNase H concentration optimization
  • Figure 4E is a schematic diagram of the reaction buffer optimization results
  • Figure 4F is a schematic diagram of the detected reaction temperature optimization results
  • Figure 4G is a schematic diagram of the optimization results of the volume ratio of the amplification system and the detection system
  • Figure 4H is a schematic diagram of the optimization results of fluorescent probes
  • Figure 4I is a schematic diagram of the results of detecting the lowest copy number of RNA before and after optimization of the reaction system.
  • Figure 5 is a schematic diagram of the efficiency of the optimized reaction system in detecting clinical samples (SARS-CoV-2, A and B).
  • Figure 6A- Figure 6E are schematic diagrams of the optimization results of the reaction system without nucleic acid extraction:
  • Figure 6A is a schematic diagram of screening of pathogenic nucleic acid cleavage methods
  • Figure 6B is a schematic diagram of the optimization results of the ratio of pathogen transfer solution and lysis solution
  • Figure 6C is a schematic diagram of the optimization results of pyrolysis temperature
  • Figure 6D is a schematic diagram of the limit of detection (LOD) results of the SARS-CoV-2 pseudovirus assay reaction
  • Figure 6E is a schematic diagram of the detection limit results of influenza A (IAV) compared with RT-qPCR.
  • Figure 7 is a schematic diagram of the positive coincidence rate results of the CRISPR-Cas12a reaction system that does not require nucleic acid extraction and does not require opening the cap when detecting IAV-positive samples (all samples with Ct values less than 36) and RT-qPCR technology.
  • SARS-CoV-2 samples were collected by the Shanghai Customs Port Clinic. Influenza A virus (IAV), influenza B virus (IBV) and negative samples were collected by Ruijin Hospital. All samples are first used for clinical molecular diagnosis, and excess samples are stored for further research. No personally identifiable information is collected. The study was approved by the ethics committees of ShanghaiTech University and Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine.
  • IAV Influenza A virus
  • IBV influenza B virus
  • negative samples were collected by Ruijin Hospital. All samples are first used for clinical molecular diagnosis, and excess samples are stored for further research. No personally identifiable information is collected. The study was approved by the ethics committees of ShanghaiTech University and Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine.
  • RNA was extracted from nasopharyngeal (NP) swab virus transfer medium (VTM) (Yocon Biology, Beijing, China) using the TIANamp Virus DNA/RNA Kit (TIANGEN, Beijing, China) according to the product instructions.
  • NP nasopharyngeal
  • VTM virus transfer medium
  • RT-qPCR was performed in a QuantStudio 6 Flex System thermocycler (Applied Biosystems, USA) using the One Step PrimeScript RT-PCR Kit (Takara, China). Primers were designed according to the instructions of the Chinese Center for Disease Control (CDC).
  • the cycling conditions of the reaction were as follows: reverse transcription reaction at 42°C for 5 min, heat activation at 95°C for 10 s and 40 cycles of a denaturation step at 95°C for 5 s followed by an annealing and extension step at 60°C for 34 s.
  • the inventor designed RT-RPA primers targeting the N gene of SARS-CoV-2 (as shown in Table 1). Different forward and reverse primer combinations were used to amplify the RNA of SARS-CoV-2, and the best primer pairs were screened through Cas12a fluorescence reaction verification. The primer pair with the best performance was then selected for subsequent RT-RPA reactions.
  • RNA targets To generate RNA targets, the target sequences of the IAV-M, IBV-HA and SARS-CoV-2-N genes were cloned into the PUC57 plasmid and then PCR amplified using primers containing the T7 promoter. The PCR product was confirmed by gel electrophoresis and used as IVT template (in vitro transcription template) after purification by gel extraction kit (Omega, USA). The IVT template of crRNA (shown in Table 2) and target RNA was transcribed overnight at 37°C using HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB, USA).
  • RNA copy number (copy number/ ⁇ L) [6.02 ⁇ 10 23 ⁇ RNA concentration (ng/ ⁇ L) ⁇ 10 -9 ]/(full transcript This length ⁇ 330).
  • RT-RPA was performed using a commercial kit (WLRB8207KIT, AmpFuture, China) according to the product instructions. Briefly, a 25 ⁇ L reaction containing 14.7 ⁇ L of rehydration buffer, 5 ⁇ L of RNA sample to be tested, 0.4 ⁇ M of each primer, and 14 mM magnesium acetate was incubated at 42°C for 30 min.
  • reaction mixture contains 5 ⁇ L RT-RPA product, 2 ⁇ L 10 ⁇ Buffer 3.1 (NEB, B7203S), 100nM crRNA, 50nM LbCas12a and 1.25 ⁇ M single-stranded DNA (ssDNA) reporter probe, at 37 Incubate at °C for 30 minutes. Fluorescence signals were monitored using a SpectraMax iD3 multi-mode microplate reader ( ⁇ ex: 485nm; ⁇ em: 550nm).
  • Figure 4A is a schematic diagram of the reaction system and process design that requires no extraction and no need to open the lid.
  • Figure 4B- Figure 4H based on the contamination-free detection method in closed-tube reactions, we optimized various reaction conditions for this assay.
  • Figure 4B and Figure 4C in order to optimize the reaction time, we added RT-RPA products with different reaction times (5, 10, 15, and 20 minutes) in the Cas12a-based assay and measured the kinetics of the fluorescence signal.
  • Figure 4D for RT-RPA optimization, an isothermal amplification reaction was performed by adding different concentrations of ribonuclease H (RNase H) (0, 0.1, 0.2, 0.5, and 1.0 U/ ⁇ L).
  • RNase H ribonuclease H
  • RT-RPA kit 100nM crRNA, 50nM LbCas12a and 1.25 ⁇ M ssDNA reporter probe. Optimization was performed in an iterative manner, modifying only one reagent per experiment. The optimal choice for each reaction condition is based on better fluorescence kinetics or lower LOD as optimal detection performance. When an optimal reaction condition is determined, the previous reaction condition will be replaced and integrated into the plan for optimization of the next reaction condition.
  • the optimized reaction conditions were as follows: (1) In the RT-RPA reaction step, add 0.1 U/ ⁇ L RNase H to 25 ⁇ L reaction mixture, and then incubate at 42°C for 15 min. (2) Then optimize the Cas12a-mediated detection step.
  • 100 ⁇ L reaction system contains 25 ⁇ L of RT-RPA product, 1 ⁇ optimized reaction buffer (pH 7.9, 10mM Tris-HCl, 20mM NaCl, 15mM MgCl 2 , 0.5mM Dithiothreitol, 100 ⁇ g/mL bovine serum albumin, 100 nM crRNA, 50 nM LbCas12a and 1.25 ⁇ M 5C-FQ reporter probe, incubated at 42°C for 10 minutes.
  • 1 ⁇ optimized reaction buffer pH 7.9, 10mM Tris-HCl, 20mM NaCl, 15mM MgCl 2 , 0.5mM Dithiothreitol, 100 ⁇ g/mL bovine serum albumin, 100 nM crRNA, 50 nM LbCas12a and 1.25 ⁇ M 5C-FQ reporter probe, incubated at 42°C for 10 minutes.
  • the LOD before and after optimization is shown in Figure 4I. As can be seen from the figure, the above method reduces the detection limit LOD from 100 copies/ ⁇ L to 1 copy/ ⁇ L.
  • lentivirus containing the N gene fragment of SARS-CoV-2 (Beyotime, China) was incorporated into VTM to simulate clinical samples.
  • lysis buffer volume optimization samples were diluted in optimal lysis buffer at volume ratios of 1:1, 1:5, and 1:10.
  • cleavage temperature optimization cleavage reactions were performed at 37, 65, and 95°C. The results are shown in Figures 6A to 6E.

Abstract

Disclosed is a CRISPR-based detection kit and use thereof. The detection kit of the present invention comprises a gene editing system. The gene editing system comprises a nuclease and a guide RNA. The sequence of the guide RNA is selected from one or more of nucleotide sequences set forth in SEQ ID NOs: 1-4. The kit of the present invention can reduce the detection limit while improving the detection efficiency within a shortened reaction time and reduced reaction temperature. The operation does not include nucleic acid extraction, eliminating the need for uncovering, thus being simple, convenient, and rapid.

Description

基于CRISPR的核酸检测试剂盒及其应用CRISPR-based nucleic acid detection kits and their applications
本申请要求申请日为2022/4/24的中国专利申请2022104359963的优先权。本申请引用上述中国专利申请的全文。This application claims the priority of Chinese patent application 2022104359963 with a filing date of 2022/4/24. This application cites the full text of the above-mentioned Chinese patent application.
技术领域Technical field
本发明属于生物技术领域，具体涉及一种免抽提、免开盖的基于CRISPR的核酸检测试剂盒及其应用。The invention belongs to the field of biotechnology, and specifically relates to an extraction-free and cap-opening CRISPR-based nucleic acid detection kit and its application.
背景技术Background technique
引起COVID-19的病原已经被确认为是β属冠状病毒，被命名为SARS-CoV-2。和其他冠状病毒类似，SARS-CoV-2是单链、正义的RNA病毒。SARS-CoV-2和SARS-CoV以及中东呼吸综合症冠状病毒(MERS-CoV)分别具有79％和50％的核酸相似性。针对COVID-19的现有分子诊断方法主要是基于反转录PCR(RT-PCR)。SARS-CoV-2的E、N以及ORF1ab基因是常用的RT-PCR的靶标基因。高通量的RT-PCR平台也已被研发出来用于大规模的诊断。不过，RT-PCR通常依赖于特别的仪器，因此大规模的RT-PCR检测往往局限于在医院的病人。同时，目前疫情防控要求为48小时内核酸检测结果，而缺乏对高危环境(如机场、高铁等公共场所)的检测手段，亦为疫情防控带来极大的不确定性。The pathogen causing COVID-19 has been confirmed to be a beta coronavirus, named SARS-CoV-2. Similar to other coronaviruses, SARS-CoV-2 is a single-stranded, positive-sense RNA virus. SARS-CoV-2 and SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) share 79% and 50% nucleic acid similarity, respectively. Existing molecular diagnostic methods for COVID-19 are mainly based on reverse transcription PCR (RT-PCR). The E, N and ORF1ab genes of SARS-CoV-2 are commonly used RT-PCR target genes. High-throughput RT-PCR platforms have also been developed for large-scale diagnosis. However, RT-PCR usually relies on special equipment, so large-scale RT-PCR testing is often limited to patients in hospitals. At the same time, the current epidemic prevention and control requirements are nucleic acid test results within 48 hours, and the lack of testing methods for high-risk environments (such as airports, high-speed rail and other public places) also brings great uncertainty to epidemic prevention and control.
最近研究表明，Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)技术可用于病原的低成本、便携式的分子检测。CRISPR以及CRISPR-associated genes(Cas)基因是细菌抵御外源病毒感染的免疫系统。CRISPR的模块特性使得此技术被广泛应用于基因组工程中。基于CRISPR的病原核酸的分子诊断依赖于Cas核酸酶的RNA或DNA的靶向活性，可在30分钟内实现新冠病毒的快速、准确检测。不过，目前CRISPR仍未在大范围推广使用，其中一个主要局限在于CRISPR的操作过于复杂。Recent studies have shown that Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology can be used for low-cost, portable molecular detection of pathogens. CRISPR and CRISPR-associated genes (Cas) genes are the immune system of bacteria to resist foreign viral infections. The modular nature of CRISPR makes this technology widely used in genome engineering. CRISPR-based molecular diagnosis of pathogenic nucleic acids relies on the RNA or DNA targeting activity of Cas nuclease, which can achieve rapid and accurate detection of the new coronavirus within 30 minutes. However, CRISPR has not yet been widely used. One of the main limitations is that the operation of CRISPR is too complex.
发明内容Contents of the invention
本发明所要解决的技术问题是为了克服现有技术缺少操作简便、准确快速的CRISPR反应体系，提供了一种基于CRISPR的核酸检测试剂盒及其应用。本发明的核酸检测试剂盒能够快速准确地反映CRISPR的检测结果，例如在检测SARS-CoV-2时利用特定crRNA，高灵敏度、高特异性识别病毒基因；并可免核酸抽提、免开盖进行检测，降低了污染可能性和假阳性率，具有较高的应用价值。 The technical problem to be solved by the present invention is to overcome the lack of an easy-to-operate, accurate and fast CRISPR reaction system in the existing technology, and provide a CRISPR-based nucleic acid detection kit and its application. The nucleic acid detection kit of the present invention can quickly and accurately reflect the detection results of CRISPR. For example, when detecting SARS-CoV-2, specific crRNA is used to identify viral genes with high sensitivity and specificity; and it can eliminate the need for nucleic acid extraction and opening of the lid. Detection reduces the possibility of contamination and false positive rate, and has high application value.
本发明通过以下技术方案解决上述技术问题。The present invention solves the above technical problems through the following technical solutions.
本发明的第一方面提供一种基因编辑系统，所述基因编辑系统包括核酸酶和引导RNA；其中，所述引导RNA的序列选自如SEQ ID NO:1～4所示的核苷酸序列中的一种或多种。A first aspect of the present invention provides a gene editing system, which includes a nuclease and a guide RNA; wherein, the sequence of the guide RNA is selected from the nucleotide sequences shown in SEQ ID NO: 1 to 4 of one or more.
本发明一些实施方案中，所述引导RNA的序列如SEQ ID NO:1～4任一项所示。In some embodiments of the present invention, the sequence of the guide RNA is as shown in any one of SEQ ID NO: 1 to 4.
本发明中，所述核酸酶可为本领域常规用于破坏核酸中的磷酸二酯键以产生核酸链的断裂的酶，优选地为Cas蛋白。In the present invention, the nuclease may be an enzyme conventionally used in the art to destroy phosphodiester bonds in nucleic acids to cause cleavage of nucleic acid chains, and is preferably Cas protein.
本发明一些实施方案中，所述Cas蛋白为Cas12a或Cas13a；In some embodiments of the present invention, the Cas protein is Cas12a or Cas13a;
本发明中，当所述Cas蛋白为Cas12a时，所述Cas12a优选AsCas12a(来自Acidaminococcus sp.BV3L6)、BbCas12a(来自Beauveria bassiana KA00251)、BoCas12a(来自Bacteroidetes oral)、FnCas12a(来自Francisella novicida U112)、HkCas12a(来自Helcococcus kunzii)、Lb4Cas12a(来自Lachnospiraceae bacterium MC2017)、Lb5Cas12a(来自Lachnospiraceae bacterium NC2008)、LbCas12a(来自Lachnospiraceae bacterium ND2006)、OsCas12a(来自Oribacterium sp.)或TsCas12a(来自Thiomicrospira sp.XS5)。In the present invention, when the Cas protein is Cas12a, the Cas12a is preferably AsCas12a (from Acidaminococcus sp.BV3L6), BbCas12a (from Beauveria bassiana KA00251), BoCas12a (from Bacteroidetes oral), FnCas12a (from Francisella novicida U112), HkCas12a (from Helcococcus kunzii), Lb4Cas12a (from Lachnospiraceae bacterium MC2017), Lb5Cas12a (from Lachnospiraceae bacterium NC2008), LbCas12a (from Lachnospiraceae bacterium ND2006), OsCas12a (from Oribacterium sp.) or TsCas12a (from Thiomicrospira sp.XS5).
本发明的第二方面提供一种分离的核酸，所述核酸编码如第一方面所述的基因编辑系统。A second aspect of the invention provides an isolated nucleic acid encoding the gene editing system of the first aspect.
本发明的第三方面提供一种用于核酸检测的检测体系，所述检测体系包括基因编辑系统和反应缓冲液；其中，所述基因编辑系统包括核酸酶和引导RNA，所述反应缓冲液包括10～50mM的NaCl、5～50mM的MgCl2、5～50mM的Tris-HCl、0.1～1.0mM的二硫苏糖醇和50～200μg/mL的牛血清白蛋白，pH 7～8。A third aspect of the present invention provides a detection system for nucleic acid detection. The detection system includes a gene editing system and a reaction buffer; wherein the gene editing system includes a nuclease and a guide RNA, and the reaction buffer includes 10～50mM NaCl, 5～50mM MgCl 2 , 5～50mM Tris-HCl, 0.1～1.0mM dithiothreitol and 50～200μg/mL bovine serum albumin, pH 7～8.
本发明中，所述核酸酶如第一方面所述，所述引导RNA为本领域常规能够结合所述核酸酶并将所述核酸酶引导至目的基因的RNA，例如为sgRNA或crRNA；所述目的基因可为病毒基因，例如RNA病毒的基因。In the present invention, the nuclease is as described in the first aspect, and the guide RNA is a conventional RNA in the art that can bind the nuclease and guide the nuclease to the target gene, such as sgRNA or crRNA; The gene of interest may be a viral gene, such as a gene of an RNA virus.
本发明一些实施方案中，所述基因编辑系统为如第一方面所述的基因编辑系统。In some embodiments of the present invention, the gene editing system is the gene editing system described in the first aspect.
本发明中，所述引导RNA和所述核酸酶的工作浓度可为本领域常规，例如所述引导RNA的工作浓度为50～200nM，例如100nM；所述核酸酶的工作浓度为25～100nM，例如50nM。In the present invention, the working concentration of the guide RNA and the nuclease can be conventional in the art, for example, the working concentration of the guide RNA is 50-200 nM, such as 100 nM; the working concentration of the nuclease is 25-100 nM, For example 50nM.
本发明一些实施方案中，所述反应缓冲液包括20～30mM的NaCl、15～25mM的MgCl2、10～20mM的Tris-HCl、0.5～0.75mM的二硫苏糖醇和100～200μg/mL的牛血清白蛋白，pH 7～8。In some embodiments of the present invention, the reaction buffer includes 20-30mM NaCl, 15-25mM MgCl 2 , 10-20mM Tris-HCl, 0.5-0.75mM dithiothreitol and 100-200μg/mL Bovine serum albumin, pH 7-8.
本发明一些具体实施方案中，所述反应缓冲液包括20mM的NaCl、15mM的MgCl2、 10mM的Tris-HCl、0.5mM的二硫苏糖醇和100μg/mL的牛血清白蛋白，pH7.9。In some specific embodiments of the present invention, the reaction buffer includes 20mM NaCl, 15mM MgCl 2 , 10mM Tris-HCl, 0.5mM dithiothreitol and 100μg/mL bovine serum albumin, pH 7.9.
本发明的第四方面提供一种用于核酸扩增的扩增体系，所述扩增体系包括引物、RNase H和RPA酶；其中，所述引物用于扩增待测样品中的核酸，所述RPA酶为用于重组酶聚合酶扩增的酶，所述RNase H的浓度不高于1U/μL。The fourth aspect of the present invention provides an amplification system for nucleic acid amplification. The amplification system includes primers, RNase H and RPA enzyme; wherein, the primers are used to amplify nucleic acids in the sample to be tested, so The RPA enzyme is an enzyme used for recombinase polymerase amplification, and the concentration of RNase H is not higher than 1U/μL.
本发明中，所述RPA酶可为本领域常规用于重组酶聚合酶扩增(Recombinase Polymerase Amplification，RPA)的酶，包括能够结合单链核酸引物的重组酶、单链DNA结合蛋白(ssDNA binding protein，SSB)和链置换DNA聚合酶。In the present invention, the RPA enzyme can be an enzyme conventionally used in the field for recombinase polymerase amplification (RPA), including recombinase capable of binding single-stranded nucleic acid primers, single-stranded DNA binding protein (ssDNA binding) protein, SSB) and strand-displacement DNA polymerase.
本发明一些实施方案中，所述RNase H的浓度为0.1～0.5U/μL。In some embodiments of the present invention, the concentration of RNase H is 0.1～0.5U/μL.
本发明一些实施方案中，所述引物为用于扩增SARS-CoV-2的核酸的引物；优选地为用于扩增SARS-CoV-2的N基因的引物。In some embodiments of the present invention, the primer is a primer used to amplify the nucleic acid of SARS-CoV-2; preferably, it is a primer used to amplify the N gene of SARS-CoV-2.
所述引物包括正向引物和反向引物；所述正向引物优选选自如SEQ ID NO:5～8所示的核苷酸序列中的一种或多种，所述反向引物优选选自如SEQ ID NO:9～12所示的核苷酸序列中的一种或多种。The primers include a forward primer and a reverse primer; the forward primer is preferably selected from one or more of the nucleotide sequences shown in SEQ ID NO: 5 to 8, and the reverse primer is preferably selected from the group consisting of One or more of the nucleotide sequences shown in SEQ ID NO:9~12.
本发明一些具体实施方案中，所述正向引物的核苷酸序列如SEQ ID NO:5所示，所述反向引物的核苷酸序列如SEQ ID NO:9所示；或者，所述正向引物的核苷酸序列如SEQ ID NO:6所示，所述反向引物的核苷酸序列如SEQ ID NO:10所示；或者，所述正向引物的核苷酸序列如SEQ ID NO:7所示，所述反向引物的核苷酸序列如SEQ ID NO:11所示；或者，所述正向引物的核苷酸序列如SEQ ID NO:8所示，所述反向引物的核苷酸序列如SEQ ID NO:12所示。In some specific embodiments of the present invention, the nucleotide sequence of the forward primer is as shown in SEQ ID NO:5, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO:9; or, the The nucleotide sequence of the forward primer is shown in SEQ ID NO: 6, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 10; or, the nucleotide sequence of the forward primer is shown in SEQ ID NO:7, the nucleotide sequence of the reverse primer is shown in SEQ ID NO:11; or, the nucleotide sequence of the forward primer is shown in SEQ ID NO:8, the reverse primer The nucleotide sequence of the primer is shown in SEQ ID NO:12.
本发明中，所述引物的工作浓度可为本领域常规，优选地为0.2～2μM，例如0.4μM。In the present invention, the working concentration of the primer can be conventional in the art, preferably 0.2-2 μM, such as 0.4 μM.
本发明一些实施方案中，所述扩增体系还包括逆转录酶；当待测核酸为RNA时，所述逆转录酶用于将RNA逆转录为DNA。In some embodiments of the present invention, the amplification system further includes a reverse transcriptase; when the nucleic acid to be tested is RNA, the reverse transcriptase is used to reverse transcribe RNA into DNA.
本发明的第五方面提供一种用于病原裂解的裂解体系，所述裂解体系包括病原转移液和裂解液；所述病原转移液用于维持待测样品的病原活性，所述裂解液用于暴露所述待测样品中的病原核酸；其中，所述病原转移液与所述裂解液的体积比为1:(0.5～10)。The fifth aspect of the present invention provides a lysis system for pathogen lysis. The lysis system includes a pathogen transfer solution and a lysis solution; the pathogen transfer solution is used to maintain the pathogenic activity of the sample to be tested, and the lysis solution is used to The pathogenic nucleic acid in the sample to be tested is exposed; wherein, the volume ratio of the pathogen transfer solution and the lysis solution is 1: (0.5-10).
本发明中，所述病原转移液和裂解液为本领域常规，例如所述病原转移液为北京友康，MT0301的病原转移液，所述裂解液为Epicentre，QE09050的裂解液。In the present invention, the pathogen transfer solution and lysis solution are conventional in this field. For example, the pathogen transfer solution is the pathogen transfer solution of Beijing Youkang, MT0301, and the lysis solution is the lysis solution of Epicentre, QE09050.
本发明一些实施方案中，所述病原转移液与所述裂解液的体积比为1:(1～5)；优选地为1:1。In some embodiments of the present invention, the volume ratio of the pathogen transfer solution and the lysis solution is 1: (1-5); preferably, it is 1:1.
本发明中，所述病原可为本领域常规致病生物体，优选选自病原性真菌、病毒和病原性原核生物；所述病原性原核生物优选包括细菌、支原体和衣原体； In the present invention, the pathogen can be a conventional pathogenic organism in the field, preferably selected from pathogenic fungi, viruses and pathogenic prokaryotes; the pathogenic prokaryotes preferably include bacteria, mycoplasma and chlamydia;
本发明一些实施方案中，所述病原为病毒，优选地为RNA病毒，例如SARS-CoV-2、IAV(甲型流感病毒)或IBV(乙型流感病毒)。In some embodiments of the present invention, the pathogen is a virus, preferably an RNA virus, such as SARS-CoV-2, IAV (Influenza A virus) or IBV (Influenza B virus).
本发明的第六方面提供一种用于核酸检测的试剂盒，所述试剂盒包括如第三方面所述的检测体系。A sixth aspect of the present invention provides a kit for nucleic acid detection, which kit includes the detection system as described in the third aspect.
本发明一些实施方案中，所述检测体系中的基因编辑系统如第一方面所述。In some embodiments of the present invention, the gene editing system in the detection system is as described in the first aspect.
本发明一些实施方案中，所述检测体系还包括核酸探针。In some embodiments of the present invention, the detection system further includes a nucleic acid probe.
本发明中，所述核酸探针为本领域常规以核酸序列为骨架的探针，优选地为荧光标记的单链DNA(ssDNA)。所述荧光标记是指在所述单链DNA两端分别连接发光基团和淬灭基团。例如，当所述单链DNA的序列为CCCCC时，所述发光基团可为FAM，所述淬灭基团可为BHQ1。In the present invention, the nucleic acid probe is a probe with a nucleic acid sequence as a backbone conventionally used in this field, and is preferably a fluorescently labeled single-stranded DNA (ssDNA). The fluorescent label refers to connecting a luminescent group and a quenching group to both ends of the single-stranded DNA respectively. For example, when the sequence of the single-stranded DNA is CCCCC, the luminescent group can be FAM, and the quenching group can be BHQ1.
本发明一些实施方案中，所述试剂盒还包括扩增体系，所述扩增体系包括扩增引物和RPA酶；其中，所述扩增引物用于扩增待测样品中的核酸，所述RPA酶为用于重组酶聚合酶扩增的酶；所述扩增体系优选地如第四方面所述。In some embodiments of the present invention, the kit further includes an amplification system, which includes an amplification primer and an RPA enzyme; wherein the amplification primer is used to amplify the nucleic acid in the sample to be tested, and the amplification system RPA enzyme is an enzyme used for recombinase polymerase amplification; the amplification system is preferably as described in the fourth aspect.
本发明另一些实施方案中，所述试剂盒还包括裂解体系，所述裂解体系包括病原转移液和裂解液；其中，所述病原转移液用于维持待测样品的病原活性，所述裂解液用于暴露所述待测样品中的病原核酸；所述裂解体系优选地如第五方面所述。In other embodiments of the present invention, the kit further includes a lysis system, which includes a pathogen transfer solution and a lysis solution; wherein the pathogen transfer solution is used to maintain the pathogenic activity of the sample to be tested, and the lysis solution For exposing the pathogenic nucleic acid in the sample to be tested; the lysis system is preferably as described in the fifth aspect.
本发明一些实施方案中，所述试剂盒包括检测体系、扩增体系和裂解体系，所述检测体系、扩增体系和裂解体系如上所述。In some embodiments of the present invention, the kit includes a detection system, an amplification system and a lysis system, and the detection system, amplification system and lysis system are as described above.
本发明的第七方面提供一种用于检测SARS-CoV-2的crRNA，所述crRNA为检测SARS-CoV-2的N基因的crRNA；其中，所述crRNA选自如SEQ ID NO:1～4所示的核苷酸序列中的一种或多种。The seventh aspect of the present invention provides a crRNA for detecting SARS-CoV-2, the crRNA is a crRNA for detecting the N gene of SARS-CoV-2; wherein, the crRNA is selected from the group consisting of SEQ ID NO: 1 to 4 One or more of the nucleotide sequences shown.
本发明一些实施方案中，所述crRNA为如SEQ ID NO:1～4任一项所示的核苷酸序列。In some embodiments of the present invention, the crRNA is a nucleotide sequence as shown in any one of SEQ ID NO: 1 to 4.
本发明的第八方面提供一种核酸检测的方法，所述方法包括：使待测样品与如第三方面所述的检测体系发生反应，收集所述反应产生的荧光信号。An eighth aspect of the present invention provides a nucleic acid detection method, which method includes: reacting a sample to be tested with the detection system as described in the third aspect, and collecting the fluorescence signal generated by the reaction.
本发明一些实施方案中，所述反应的时间为5～25min；例如10～25min、10～20min或15～20min。In some embodiments of the present invention, the reaction time is 5 to 25 minutes; for example, 10 to 25 minutes, 10 to 20 minutes, or 15 to 20 minutes.
本发明一些实施方案中，所述反应的温度为35～45℃；例如35～42℃、37～42℃或39～42℃。In some embodiments of the present invention, the reaction temperature is 35-45°C; for example, 35-42°C, 37-42°C or 39-42°C.
本发明一些实施方案中，所述方法还包括在进行所述反应之前，所述待测样品中的核酸在如第四方面所述的扩增体系中发生扩增。 In some embodiments of the present invention, the method further includes amplifying the nucleic acid in the sample to be tested in the amplification system as described in the fourth aspect before performing the reaction.
本发明一些实施方案中，所述方法还包括在进行所述反应之前，所述待测样品使用如第五方面所述的裂解体系进行裂解，以暴露所述待测样品中的核酸，以实现免核酸抽提检测。In some embodiments of the present invention, the method further includes, before performing the reaction, the sample to be tested is lysed using the cleavage system as described in the fifth aspect to expose the nucleic acid in the sample to be tested, so as to achieve Nucleic acid extraction-free testing.
本发明一些实施方案中，所述方法还包括在进行所述反应之前，所述待测样品中的核酸在如第四方面所述的扩增体系中发生扩增；并在扩增反应之前，所述待测样品与如第五方面所述的裂解体系进行裂解，以暴露所述待测样品中的核酸。In some embodiments of the present invention, the method further includes, before performing the reaction, the nucleic acid in the sample to be tested is amplified in the amplification system as described in the fourth aspect; and before the amplification reaction, The sample to be tested is lysed with the cleavage system as described in the fifth aspect to expose the nucleic acid in the sample to be tested.
本发明中，所述扩增体系与所述检测体系的反应体积比为1:(1～5)；优选地为1:(2～4)，例如为1:3。In the present invention, the reaction volume ratio of the amplification system and the detection system is 1:(1-5); preferably 1:(2-4), for example, 1:3.
本发明中，所述扩增体系预存于发生所述检测反应的封闭容器内。In the present invention, the amplification system is pre-stored in a closed container where the detection reaction occurs.
本发明一些实施方案中，所述检测体系在所述扩增结束后加入优选通过注射加入所述封闭容器内，以实现免开盖检测。In some embodiments of the present invention, the detection system is added into the closed container after the amplification is completed, preferably by injection, to achieve detection without opening the lid.
本发明中，所述裂解的温度为37℃～95℃，例如为65℃～95℃。In the present invention, the pyrolysis temperature is 37°C to 95°C, for example, 65°C to 95°C.
本发明的第九方面提供一种如第一方面所述的基因编辑系统、如第二方面所述的核酸、如第三方面所述的检测体系、如第四方面所述的扩增体系、如第五方面所述的裂解体系、如第六方面所述的试剂盒或者如第七方面所述的crRNA在制备检测病原的核酸的试剂中的应用。A ninth aspect of the present invention provides a gene editing system as described in the first aspect, a nucleic acid as described in the second aspect, a detection system as described in the third aspect, an amplification system as described in the fourth aspect, Application of the lysis system as described in the fifth aspect, the kit as described in the sixth aspect, or the crRNA as described in the seventh aspect in preparing reagents for detecting pathogenic nucleic acids.
本发明中，所述病原如第五方面所述。In the present invention, the pathogen is as described in the fifth aspect.
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。On the basis of common sense in the field, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.
本发明所用试剂和原料均市售可得。The reagents and raw materials used in the present invention are all commercially available.
本发明的积极进步效果在于：The positive progressive effects of the present invention are:
(1)本发明的试剂盒使用靶向SARS-CoV-2的N基因的crRNA，能够在RNA病毒中高灵敏度、高特异性识别并切割该基因(在优选的实施例中LOD从100copies/μL降低为1copy/μL)，并能够广谱检测SARS-CoV-2的野生型及各突变株；(1) The kit of the present invention uses crRNA targeting the N gene of SARS-CoV-2 and can recognize and cleave this gene in RNA viruses with high sensitivity and specificity (in the preferred embodiment, the LOD is reduced from 100 copies/μL (1copy/μL), and can detect wild-type and mutant strains of SARS-CoV-2 in a broad spectrum;
(2)本发明优化的反应体系能够在缩短的反应时间、降低的反应温度内提高检测效率的同时降低检测限(可达1copy/μL)，假阳性率低；(2) The optimized reaction system of the present invention can improve detection efficiency while reducing detection limit (up to 1 copy/μL) within shortened reaction time and reduced reaction temperature, and has a low false positive rate;
(3)使用本发明的试剂盒检测RNA病毒，灵敏度可达95.92％，最高阳性符合率可达95.56％，最高阴性符合率可达100％，具有优异的检测效率。(3) Using the kit of the present invention to detect RNA viruses, the sensitivity can reach 95.92%, the highest positive coincidence rate can reach 95.56%, and the highest negative coincidence rate can reach 100%, with excellent detection efficiency.
(4)本发明的试剂盒能够实现连续、密闭(免开盖)的闭管检测，通过优化反应体系实现各反应的兼容；并能够实现免核酸抽提进行核酸检测，具有较高的应用价值。 (4) The kit of the present invention can realize continuous, airtight (no need to open the lid) closed-tube detection, achieve compatibility of various reactions by optimizing the reaction system, and can realize nucleic acid detection without nucleic acid extraction, which has high application value. .
附图说明Description of the drawings
图1为本发明针对SARS-CoV-2的N基因的crRNA序列优化结果示意图。Figure 1 is a schematic diagram of the crRNA sequence optimization results of the present invention for the N gene of SARS-CoV-2.
图2为本发明针对SARS-CoV-2的N基因的优化crRNA和引物特异性检测SARS-CoV-2的结果示意图。Figure 2 is a schematic diagram of the results of the specific detection of SARS-CoV-2 using optimized crRNA and primers for the N gene of SARS-CoV-2 according to the present invention.
图3为本发明针对SARS-CoV-2的N基因的优化crRNA和引物广谱检测SARS-CoV-2的野生型及各个突变株的结果示意图。Figure 3 is a schematic diagram of the results of broad-spectrum detection of SARS-CoV-2 wild type and various mutant strains using optimized crRNA and primers for the N gene of SARS-CoV-2 according to the present invention.
图4A-图4I为本发明方法的优化示意图：Figure 4A-Figure 4I are optimization schematic diagrams of the method of the present invention:
图4A为免抽提、免开盖的反应体系和流程设计示意图；Figure 4A is a schematic diagram of the reaction system and process design that requires no extraction and no need to open the lid;
图4B为扩增时间优化结果示意图；Figure 4B is a schematic diagram of the amplification time optimization results;
图4C为检测的反应时间优化结果示意图；Figure 4C is a schematic diagram of the detection reaction time optimization results;
图4D为RNase H浓度优化结果示意图；Figure 4D is a schematic diagram of the results of RNase H concentration optimization;
图4E为反应缓冲液优化结果示意图；Figure 4E is a schematic diagram of the reaction buffer optimization results;
图4F为检测的反应温度优化结果示意图；Figure 4F is a schematic diagram of the detected reaction temperature optimization results;
图4G为扩增体系与检测体系的体积比优化结果示意图；Figure 4G is a schematic diagram of the optimization results of the volume ratio of the amplification system and the detection system;
图4H为荧光探针优化结果示意图；Figure 4H is a schematic diagram of the optimization results of fluorescent probes;
图4I为反应体系优化前后检测RNA最低拷贝数的结果示意图。Figure 4I is a schematic diagram of the results of detecting the lowest copy number of RNA before and after optimization of the reaction system.
图5为优化后的反应体系检测临床样本(SARS-CoV-2、甲流和乙流)的效率示意图。Figure 5 is a schematic diagram of the efficiency of the optimized reaction system in detecting clinical samples (SARS-CoV-2, A and B).
图6A-图6E为免核酸抽提的反应体系的优化结果示意图：Figure 6A-Figure 6E are schematic diagrams of the optimization results of the reaction system without nucleic acid extraction:
图6A为病原核酸裂解方式筛选示意图；Figure 6A is a schematic diagram of screening of pathogenic nucleic acid cleavage methods;
图6B为病原转移液和裂解液比例的优化结果示意图；Figure 6B is a schematic diagram of the optimization results of the ratio of pathogen transfer solution and lysis solution;
图6C为裂解温度的优化结果示意图；Figure 6C is a schematic diagram of the optimization results of pyrolysis temperature;
图6D为利用SARS-CoV-2假病毒测定反应的检测极限(LOD)结果示意图；Figure 6D is a schematic diagram of the limit of detection (LOD) results of the SARS-CoV-2 pseudovirus assay reaction;
图6E为与RT-qPCR比较，检测甲流(IAV)的检测限结果示意图。Figure 6E is a schematic diagram of the detection limit results of influenza A (IAV) compared with RT-qPCR.
图7为免核酸抽提、免开盖的CRISPR-Cas12a反应体系检测IAV阳性样本(均为Ct值小于36的样本)时与RT-qPCR技术的阳性符合率结果示意图。Figure 7 is a schematic diagram of the positive coincidence rate results of the CRISPR-Cas12a reaction system that does not require nucleic acid extraction and does not require opening the cap when detecting IAV-positive samples (all samples with Ct values less than 36) and RT-qPCR technology.
具体实施方式Detailed ways
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。 The present invention is further described below by means of examples, but the present invention is not limited to the scope of the described examples. Experimental methods that do not indicate specific conditions in the following examples should be selected according to conventional methods and conditions, or according to product specifications.
实施例1Example 1
临床样本和伦理声明Clinical Samples and Ethics Statement
SARS-CoV-2样本由上海海关口岸门诊采集。甲型流感病毒(IAV)、乙型流感病毒(IBV)和阴性样本由瑞金医院采集。所有样本首先用于临床分子诊断，多余的样本储存起来以供进一步研究，不收集任何个人身份信息。该研究得到了上海科技大学及上海交通大学医学院附属瑞金医院伦理委员会的批准。SARS-CoV-2 samples were collected by the Shanghai Customs Port Clinic. Influenza A virus (IAV), influenza B virus (IBV) and negative samples were collected by Ruijin Hospital. All samples are first used for clinical molecular diagnosis, and excess samples are stored for further research. No personally identifiable information is collected. The study was approved by the ethics committees of ShanghaiTech University and Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.
从临床样本中提取RNA和RT-qPCR检测RNA extraction and RT-qPCR detection from clinical samples
根据产品说明书，使用TIANamp Virus DNA/RNA Kit(TIANGEN,Beijing,China)从鼻咽(NP)拭子病毒转移培养基(VTM)(Yocon Biology,Beijing,China)中提取RNA。RNA was extracted from nasopharyngeal (NP) swab virus transfer medium (VTM) (Yocon Biology, Beijing, China) using the TIANamp Virus DNA/RNA Kit (TIANGEN, Beijing, China) according to the product instructions.
使用One Step PrimeScript RT-PCR Kit(Takara，中国)在QuantStudio 6 Flex System thermocycler(Applied Biosystems，USA)中进行RT-qPCR。引物是根据中国疾病控制中心(CDC)的说明设计。RT-qPCR was performed in a QuantStudio 6 Flex System thermocycler (Applied Biosystems, USA) using the One Step PrimeScript RT-PCR Kit (Takara, China). Primers were designed according to the instructions of the Chinese Center for Disease Control (CDC).
反应的循环条件如下：逆转录反应在42℃5min，在95℃热激活化10s和40个循环在95℃5s变性步骤随后退火和延伸步骤在60℃下保持34s。The cycling conditions of the reaction were as follows: reverse transcription reaction at 42°C for 5 min, heat activation at 95°C for 10 s and 40 cycles of a denaturation step at 95°C for 5 s followed by an annealing and extension step at 60°C for 34 s.
引物设计和RT-RPA检测Primer design and RT-RPA detection
发明人设计了针对SARS-CoV-2的N基因的RT-RPA引物(如表1所示)。使用不同的正向和反向引物组合扩增SARS-CoV-2的RNA，并通过Cas12a荧光反应验证筛选最佳引物对。然后选择性能最佳的引物对用于后续的RT-RPA反应。The inventor designed RT-RPA primers targeting the N gene of SARS-CoV-2 (as shown in Table 1). Different forward and reverse primer combinations were used to amplify the RNA of SARS-CoV-2, and the best primer pairs were screened through Cas12a fluorescence reaction verification. The primer pair with the best performance was then selected for subsequent RT-RPA reactions.
表1 SARS-CoV-2的N基因的RT-RPA引物
Table 1 RT-RPA primers for the N gene of SARS-CoV-2
crRNA和RNA靶标的制备Preparation of crRNA and RNA targets
为了产生RNA靶点，将IAV-M、IBV-HA和SARS-CoV-2-N基因的靶序列克隆到PUC57质粒中，然后使用含有T7启动子的引物进行PCR扩增。PCR产物通过凝胶电泳确认，并在通过凝胶提取试剂盒(Omega,USA)纯化后用作IVT模板(体外转录模板)。使用HiScribe T7 Quick High Yield RNA Synthesis Kit(NEB，USA)在37℃下转录crRNA(如表2所示)和靶RNA的IVT模板过夜。通过添加脱氧核糖核酸酶I(DNase I)去除转录的RNA中的DNA模板，合成的RNA通过苯酚:氯仿提取然后乙醇沉淀进行纯化。纯化后的RNA浓度用Nanodrop分光光度计定量，拷贝数计算公式为：RNA拷贝数(拷贝数/μL)＝[6.02×1023×RNA浓度(ng/μL)×10-9]/(全转录本长度×330)。To generate RNA targets, the target sequences of the IAV-M, IBV-HA and SARS-CoV-2-N genes were cloned into the PUC57 plasmid and then PCR amplified using primers containing the T7 promoter. The PCR product was confirmed by gel electrophoresis and used as IVT template (in vitro transcription template) after purification by gel extraction kit (Omega, USA). The IVT template of crRNA (shown in Table 2) and target RNA was transcribed overnight at 37°C using HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB, USA). The DNA template in the transcribed RNA was removed by adding deoxyribonuclease I (DNase I), and the synthesized RNA was purified by phenol:chloroform extraction followed by ethanol precipitation. The purified RNA concentration was quantified using a Nanodrop spectrophotometer, and the copy number calculation formula was: RNA copy number (copy number/μL) = [6.02 × 10 23 × RNA concentration (ng/μL) × 10 -9 ]/(full transcript This length × 330).
表2 SARS-CoV-2的N基因的crRNA
Table 2 crRNA of N gene of SARS-CoV-2
RT-RPA和基于Cas12a的核苷酸检测优化Optimization of RT-RPA and Cas12a-based nucleotide detection
在优化之前，按照Wang et al.,2021中的描述进行了检测。该检测包括两个步骤：(1)根据产品说明书，使用商业试剂盒(WLRB8207KIT，AmpFuture，中国)进行RT-RPA。简而言之，将含有14.7μL再水化缓冲液、5μL待测RNA样品、0.4μM每种引物和14mM醋酸镁的25μL反应液在42℃下孵育30分钟。(2)对于Cas12a荧光检测，20μL反应混合物中包含5μL RT-RPA产物、2μL 10×Buffer 3.1(NEB，B7203S)、100nM crRNA、50nM LbCas12a和1.25μM单链DNA(ssDNA)报告探针，在37℃下孵育30分钟。使用SpectraMax iD3多模式酶标仪(λex：485nm；λem：550nm)监测荧光信号。Before optimization, detection was performed as described in Wang et al., 2021. The assay included two steps: (1) RT-RPA was performed using a commercial kit (WLRB8207KIT, AmpFuture, China) according to the product instructions. Briefly, a 25 μL reaction containing 14.7 μL of rehydration buffer, 5 μL of RNA sample to be tested, 0.4 μM of each primer, and 14 mM magnesium acetate was incubated at 42°C for 30 min. (2) For Cas12a fluorescence detection, 20μL reaction mixture contains 5μL RT-RPA product, 2μL 10×Buffer 3.1 (NEB, B7203S), 100nM crRNA, 50nM LbCas12a and 1.25μM single-stranded DNA (ssDNA) reporter probe, at 37 Incubate at ℃ for 30 minutes. Fluorescence signals were monitored using a SpectraMax iD3 multi-mode microplate reader (λex: 485nm; λem: 550nm).
图4A为免抽提、免开盖的反应体系和流程设计示意图。如图4B-图4H所示，基于闭管反应中的无污染检测方法，我们优化了该测定的各种反应条件。图4B和图4C中，为了优化反应时间，我们在基于Cas12a的检测中添加了不同反应时间(5、10、15和20分钟)的RT-RPA产物，并测量了荧光信号的动力学。图4D中，对于RT-RPA优化，通过添加不同浓度的核糖核酸酶H(RNase H)(0、0.1、0.2、0.5和1.0U/μL)进行等温扩增反应。图4E中，为了优化反应缓冲液，在不同浓度的Na+(0、5、10、20、50和100mM)、Mg2+(0、5、10、15、20和25mM)、Tris-HCl(0、5、10、20、50和100mM)、DTT(二硫苏糖醇)(0、0.5、1、2、5和10mM)或BSA(牛血清白蛋白)(0、50、100、200和500mM)的缓冲液中进行基于Cas12a的检测，并筛选出最佳的缓冲液(即图中 的Optimiezed Buffer)。图4F中，对于孵育温度优化，Cas12a介导的荧光反应在不同温度(35、37、39、42和45℃)下进行孵育。图4G中，对于RT-RPA产物输入量优化，在RT-RPA产物与Cas12a反应的不同体积比(1:1、1:2、1:3、1:4和1:5)下进行测定。图4H中，对于ssDNA报告探针优化，使用具有各种序列(5A-、5T-、5C-和5G-FQ报告基因)的ssDNA报告探针进行Cas12a介导的荧光反应。Figure 4A is a schematic diagram of the reaction system and process design that requires no extraction and no need to open the lid. As shown in Figure 4B-Figure 4H, based on the contamination-free detection method in closed-tube reactions, we optimized various reaction conditions for this assay. In Figure 4B and Figure 4C, in order to optimize the reaction time, we added RT-RPA products with different reaction times (5, 10, 15, and 20 minutes) in the Cas12a-based assay and measured the kinetics of the fluorescence signal. In Figure 4D, for RT-RPA optimization, an isothermal amplification reaction was performed by adding different concentrations of ribonuclease H (RNase H) (0, 0.1, 0.2, 0.5, and 1.0 U/μL). In Figure 4E, in order to optimize the reaction buffer, at different concentrations of Na + (0, 5, 10, 20, 50 and 100mM), Mg 2+ (0, 5, 10, 15, 20 and 25mM), Tris-HCl (0, 5, 10, 20, 50 and 100mM), DTT (dithiothreitol) (0, 0.5, 1, 2, 5 and 10mM) or BSA (bovine serum albumin) (0, 50, 100, 200 and 500mM) in buffers to perform Cas12a-based detection and screen out the best buffer (i.e. in the figure Optimiezed Buffer). In Figure 4F, for incubation temperature optimization, the Cas12a-mediated fluorescence reaction was incubated at different temperatures (35, 37, 39, 42, and 45°C). In Figure 4G, for the optimization of RT-RPA product input, measurements were performed at different volume ratios (1:1, 1:2, 1:3, 1:4, and 1:5) of the reaction between RT-RPA product and Cas12a. In Figure 4H, for ssDNA reporter probe optimization, Cas12a-mediated fluorescence reactions were performed using ssDNA reporter probes with various sequences (5A-, 5T-, 5C-, and 5G-FQ reporter genes).
对于所有优化实验，以下试剂均适用：For all optimization experiments, the following reagents are suitable:
RT-RPA试剂盒、100nM crRNA、50nM LbCas12a和1.25μM ssDNA报告探针。优化以迭代方式进行，每次实验仅修改一种试剂。每种反应条件的最佳选择是基于更好的荧光动力学或更低的LOD作为最佳检测性能。当一个最佳反应条件确定后，将取代前一个反应条件整合到方案中，用于下一个反应条件优化。RT-RPA kit, 100nM crRNA, 50nM LbCas12a and 1.25μM ssDNA reporter probe. Optimization was performed in an iterative manner, modifying only one reagent per experiment. The optimal choice for each reaction condition is based on better fluorescence kinetics or lower LOD as optimal detection performance. When an optimal reaction condition is determined, the previous reaction condition will be replaced and integrated into the plan for optimization of the next reaction condition.
筛选出最佳反应条件后，得到的优化的反应条件如下：(1)在RT-RPA反应步骤中，将0.1U/μL RNase H添加到25μL反应混合物中，然后在42℃孵育15min。(2)随后优化Cas12a介导的检测步骤，100μL的反应体系中含有25μL的RT-RPA产物，1×优化的反应缓冲液(pH 7.9，10mM Tris-HCl、20mM NaCl、15mM MgCl2、0.5mM二硫苏糖醇、100μg/mL牛血清白蛋白、100nM crRNA、50nM LbCas12a和1.25μM 5C-FQ报告探针，在42℃下孵育10分钟。After screening out the best reaction conditions, the optimized reaction conditions were as follows: (1) In the RT-RPA reaction step, add 0.1 U/μL RNase H to 25 μL reaction mixture, and then incubate at 42°C for 15 min. (2) Then optimize the Cas12a-mediated detection step. 100 μL reaction system contains 25 μL of RT-RPA product, 1× optimized reaction buffer (pH 7.9, 10mM Tris-HCl, 20mM NaCl, 15mM MgCl 2 , 0.5mM Dithiothreitol, 100 μg/mL bovine serum albumin, 100 nM crRNA, 50 nM LbCas12a and 1.25 μM 5C-FQ reporter probe, incubated at 42°C for 10 minutes.
优化前后的LOD如图4I所示。由图可知，上述方法将检测极限LOD从100copies/μL降低为1copy/μL。The LOD before and after optimization is shown in Figure 4I. As can be seen from the figure, the above method reduces the detection limit LOD from 100 copies/μL to 1 copy/μL.
闭管反应中的无污染检测方法Contamination-free detection method in closed-tube reactions
通过将反应溶液预装到注射器中，我们开发了一种封闭管核酸检测方法，无需重新打开盖子，以避免扩增子污染。首先，将25μL含有模板的RT-RPA反应试剂加入试管中，盖上盖子后在42℃孵育15分钟。其次，在RT-RPA反应过程中，将75μL不含扩增子的Cas12a反应混合物放入注射器中。第三，等温扩增后，在不重新打开盖子的情况下，通过注射器将Cas12a反应溶液从盖子注入管中并与RT-RPA产物混合，然后在42℃继续孵育10分钟。最后，可以通过荧光检测装置测量结果。By preloading the reaction solution into a syringe, we developed a closed-tube nucleic acid detection method that eliminates the need to reopen the cap to avoid amplicon contamination. First, add 25 μL of RT-RPA reaction reagent containing template into the test tube, cover it and incubate at 42°C for 15 minutes. Second, during the RT-RPA reaction, place 75 μL of the Cas12a reaction mixture without the amplicon into the syringe. Third, after isothermal amplification, without reopening the cap, inject the Cas12a reaction solution from the cap into the tube through a syringe and mix with the RT-RPA product, and then continue to incubate at 42°C for 10 minutes. Finally, the results can be measured via a fluorescence detection device.
RNA免提取测定RNA extraction-free assay
为了筛选最佳裂解方法，将含有SARS-CoV-2(Beyotime，中国)的N基因片段的慢病毒掺入VTM以模拟临床样本。将收集的样品与候选裂解液混合：(1)0.2％Triton X-100；(2)100mM TCEP和1mM EDTA；(3)等体积QuickExtract DNA Extraction Solution(Lucigen,USA)；仅加热(heat only)和无核酸酶水(RNase-free ddH2O)作为对照。在95℃加热5分钟后，将5μL样品用作上述基于Cas12a的闭管反应的输入，以评估裂解 效率。对于裂解缓冲液量优化，样品在最佳裂解缓冲液中以1:1、1:5和1:10的体积比稀释。对于裂解温度优化，裂解反应在37、65和95℃下进行。结果如图6A～图6E所示。To screen the best lysis method, lentivirus containing the N gene fragment of SARS-CoV-2 (Beyotime, China) was incorporated into VTM to simulate clinical samples. Mix the collected samples with the candidate lysis buffer: (1) 0.2% Triton X-100; (2) 100mM TCEP and 1mM EDTA; (3) equal volumes of QuickExtract DNA Extraction Solution (Lucigen, USA); heat only and RNase-free ddH 2 O as controls. After heating at 95°C for 5 min, 5 μL of sample was used as input to the Cas12a-based closed-tube reaction described above to assess lysis. efficiency. For lysis buffer volume optimization, samples were diluted in optimal lysis buffer at volume ratios of 1:1, 1:5, and 1:10. For cleavage temperature optimization, cleavage reactions were performed at 37, 65, and 95°C. The results are shown in Figures 6A to 6E.
统计分析Statistical Analysis
使用GraphPad Prism 8分析数据。使用Clopper-Pearson方法计算敏感性、特异性、阳性预测值(PPA)和阴性预测值(NPA)的双边置信区间。IAV阳性样本(均为Ct值小于36的样本)时与RT-qPCR技术的阳性符合率结果如图5和图7所示。由图5可知，最高阳性符合率可达95.56％，最高阴性符合率为100％。 Data were analyzed using GraphPad Prism 8. Two-sided confidence intervals for sensitivity, specificity, positive predictive value (PPA), and negative predictive value (NPA) were calculated using the Clopper-Pearson method. The positive coincidence rate results of IAV-positive samples (all samples with Ct value less than 36) and RT-qPCR technology are shown in Figure 5 and Figure 7. As can be seen from Figure 5, the highest positive coincidence rate can reach 95.56%, and the highest negative coincidence rate is 100%.

Claims (11)

  1. 一种基因编辑系统，其特征在于，所述基因编辑系统包括核酸酶和引导RNA；A gene editing system, characterized in that the gene editing system includes a nuclease and a guide RNA;
    其中，所述引导RNA的序列选自如SEQ ID NO:1～4所示的核苷酸序列中的一种或多种。Wherein, the sequence of the guide RNA is selected from one or more of the nucleotide sequences shown in SEQ ID NO: 1 to 4.
  2. 如权利要求1所述的基因编辑系统，其特征在于，所述引导RNA的序列如SEQ ID NO:1～4任一所示；和/或，The gene editing system of claim 1, wherein the sequence of the guide RNA is as shown in any one of SEQ ID NO: 1 to 4; and/or,
    所述核酸酶为Cas蛋白；The nuclease is Cas protein;
    较佳地，所述Cas蛋白为Cas12a或Cas13a；Preferably, the Cas protein is Cas12a or Cas13a;
    更佳地，所述Cas蛋白为AsCas12a、BbCas12a、BoCas12a、FnCas12a、HkCas12a、Lb4Cas12a、Lb5Cas12a、LbCas12a、OsCas12a或TsCas12a。More preferably, the Cas protein is AsCas12a, BbCas12a, BoCas12a, FnCas12a, HkCas12a, Lb4Cas12a, Lb5Cas12a, LbCas12a, OsCas12a or TsCas12a.
  3. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1或2所述的基因编辑系统。An isolated nucleic acid, characterized in that the nucleic acid encodes the gene editing system according to claim 1 or 2.
  4. 一种用于核酸检测的检测体系，其特征在于，所述检测体系包括基因编辑系统和反应缓冲液；其中，所述基因编辑系统包括核酸酶和引导RNA，所述反应缓冲液包括10～50mM的NaCl、5～50mM的MgCl2、5～50mM的Tris-HCl、0.1～1.0mM的二硫苏糖醇和50～200μg/mL的牛血清白蛋白，pH 7～8；A detection system for nucleic acid detection, characterized in that the detection system includes a gene editing system and a reaction buffer; wherein the gene editing system includes nuclease and guide RNA, and the reaction buffer includes 10-50mM NaCl, 5-50mM MgCl 2 , 5-50mM Tris-HCl, 0.1-1.0mM dithiothreitol and 50-200μg/mL bovine serum albumin, pH 7-8;
    较佳地，所述反应缓冲液包括20～30mM的NaCl、15～25mM的MgCl2、10～20mM的Tris-HCl、0.5～0.75mM的二硫苏糖醇和100～200μg/mL的牛血清白蛋白，pH 7～8；和/或，所述核酸酶为Cas蛋白；和/或，所述引导RNA的工作浓度为50～200nM例如100nM，所述核酸酶的工作浓度为25～100nM例如50nM；Preferably, the reaction buffer includes 20-30mM NaCl, 15-25mM MgCl 2 , 10-20mM Tris-HCl, 0.5-0.75mM dithiothreitol and 100-200μg/mL bovine serum albumin. Protein, pH 7-8; and/or, the nuclease is Cas protein; and/or, the working concentration of the guide RNA is 50-200 nM, such as 100 nM, and the working concentration of the nuclease is 25-100 nM, such as 50 nM ;
    更佳地，所述反应缓冲液包括20mM的NaCl、15mM的MgCl2、10mM的Tris-HCl、0.5mM的二硫苏糖醇和100μg/mL的牛血清白蛋白，pH7.9；和/或，所述Cas蛋白为Cas12a或Cas13a；所述Cas蛋白优选为AsCas12a、BbCas12a、BoCas12a、FnCas12a、HkCas12a、Lb4Cas12a、Lb5Cas12a、LbCas12a、OsCas12a或TsCas12a。More preferably, the reaction buffer includes 20mM NaCl, 15mM MgCl 2 , 10mM Tris-HCl, 0.5mM dithiothreitol and 100μg/mL bovine serum albumin, pH7.9; and/or, The Cas protein is Cas12a or Cas13a; the Cas protein is preferably AsCas12a, BbCas12a, BoCas12a, FnCas12a, HkCas12a, Lb4Cas12a, Lb5Cas12a, LbCas12a, OsCas12a or TsCas12a.
  5. 一种用于核酸扩增的扩增体系，其特征在于，所述扩增体系包括引物、RNase H和RPA酶；其中，所述引物用于扩增待测样品中的核酸，所述RPA酶为用于重组酶聚合酶扩增的酶；所述RNase H的浓度不高于1U/μL；An amplification system for nucleic acid amplification, characterized in that the amplification system includes primers, RNase H and RPA enzyme; wherein, the primers are used to amplify nucleic acids in the sample to be tested, and the RPA enzyme It is an enzyme used for recombinase polymerase amplification; the concentration of RNase H is not higher than 1U/μL;
    较佳地，所述RNase H的浓度为0.1～0.5U/μL；所述引物为用于扩增SARS-CoV-2的核酸的引物；和/或，所述扩增体系还包括逆转录酶；Preferably, the concentration of the RNase H is 0.1~0.5U/μL; the primer is a primer used to amplify the nucleic acid of SARS-CoV-2; and/or the amplification system also includes a reverse transcriptase ;
    更佳地，所述引物为用于扩增SARS-CoV-2的N基因的引物；所述引物的正向引物 优选选自如SEQ ID NO:5～8所示的核苷酸序列中的一种或多种，反向引物优选选自如SEQ ID NO:9～12所示的核苷酸序列中的一种或多种；和/或，所述引物的工作浓度为0.2～2μM例如0.4μM。More preferably, the primer is a primer used to amplify the N gene of SARS-CoV-2; the forward primer of the primer The reverse primer is preferably selected from one or more of the nucleotide sequences shown in SEQ ID NO: 5 to 8, and the reverse primer is preferably selected from one or more of the nucleotide sequences shown in SEQ ID NO: 9 to 12 or A variety of; and/or, the working concentration of the primer is 0.2-2 μM, such as 0.4 μM.
  6. 一种用于病原裂解的裂解体系，其特征在于，所述裂解体系包括病原转移液和裂解液；所述病原转移液用于维持待测样品的病原活性，所述裂解液用于暴露所述待测样品中的病原核酸；所述病原转移液优选包括0.8％氯化钠、0.04％氯化钾、0.014％氯化钙、0.02％硫酸镁(七水)、0.012％磷酸氢二钠(七水)、0.006％磷酸二氢钾、0.035％碳酸氢钠、0.1％葡萄糖和0.002％酚红钠盐；所述百分比为质量百分比；其中，所述病原转移液与所述裂解液的体积比为1:(0.5～10)；A lysis system for pathogen lysis, characterized in that the lysis system includes a pathogen transfer solution and a lysis solution; the pathogen transfer solution is used to maintain the pathogenic activity of the sample to be tested, and the lysis solution is used to expose the Pathogen nucleic acid in the sample to be tested; the pathogen transfer solution preferably includes 0.8% sodium chloride, 0.04% potassium chloride, 0.014% calcium chloride, 0.02% magnesium sulfate (heptahydrate), 0.012% disodium hydrogen phosphate (seven-water) water), 0.006% potassium dihydrogen phosphate, 0.035% sodium bicarbonate, 0.1% glucose and 0.002% phenol red sodium salt; the percentage is a mass percentage; wherein, the volume ratio of the pathogen transfer solution to the lysis solution is 1:(0.5～10);
    较佳地，所述病原转移液与所述裂解液的体积比为1:(1～5)；和/或，所述病原选自病原性真菌、病毒和病原性原核生物；所述病原性原核生物优选包括细菌、支原体和衣原体；Preferably, the volume ratio of the pathogen transfer solution to the lysis solution is 1:(1-5); and/or the pathogen is selected from pathogenic fungi, viruses and pathogenic prokaryotes; the pathogenic Prokaryotes preferably include bacteria, mycoplasma and chlamydia;
    更佳地，所述病原转移液与所述裂解液的体积比为1:1；和/或，所述病原为病毒，优选地为RNA病毒，例如SARS-CoV-2、IAV或IBV。More preferably, the volume ratio of the pathogen transfer solution to the lysis solution is 1:1; and/or the pathogen is a virus, preferably an RNA virus, such as SARS-CoV-2, IAV or IBV.
  7. 一种用于核酸检测的试剂盒，其特征在于，所述试剂盒包括如权利要求4所述的检测体系；A kit for nucleic acid detection, characterized in that the kit includes the detection system as claimed in claim 4;
    较佳地，所述基因编辑系统为如权利要求1或2所述的基因编辑系统；和/或，所述检测体系还包括核酸探针；和/或，Preferably, the gene editing system is the gene editing system according to claim 1 or 2; and/or, the detection system further includes a nucleic acid probe; and/or,
    所述试剂盒还包括扩增体系，所述扩增体系包括扩增引物和RPA酶；其中，所述扩增引物用于扩增待测样品中的核酸，所述RPA酶为用于重组酶聚合酶扩增的酶；和/或，The kit also includes an amplification system, which includes an amplification primer and an RPA enzyme; wherein the amplification primer is used to amplify the nucleic acid in the sample to be tested, and the RPA enzyme is a recombinase. Polymerase amplification enzyme; and/or,
    所述试剂盒还包括裂解体系，所述裂解体系包括病原转移液和裂解液；其中，所述病原转移液用于维持待测样品的病原活性，所述裂解液用于暴露所述待测样品中的病原核酸；The kit also includes a lysis system, which includes a pathogen transfer solution and a lysis solution; wherein, the pathogen transfer solution is used to maintain the pathogenic activity of the sample to be tested, and the lysis solution is used to expose the sample to be tested. Pathogenic nucleic acids in;
    更佳地，所述扩增体系为如权利要求5所述的扩增体系；和/或，所述裂解体系为如权利要求6所述的裂解体系；和/或，More preferably, the amplification system is the amplification system as claimed in claim 5; and/or, the lysis system is the lysis system as claimed in claim 6; and/or,
    所述核酸探针为荧光标记的单链DNA；所述荧光标记优选地包括分别位于所述单链DNA两端的发光基团和淬灭基团；所述单链DNA的序列优选地为CCCCC；例如，所述发光基团为FAM，所述淬灭基团为BHQ1。The nucleic acid probe is a fluorescently labeled single-stranded DNA; the fluorescent label preferably includes a luminescent group and a quenching group respectively located at both ends of the single-stranded DNA; the sequence of the single-stranded DNA is preferably CCCCC; For example, the luminescent group is FAM, and the quenching group is BHQ1.
  8. 一种用于检测SARS-CoV-2的crRNA，其特征在于，所述crRNA为检测SARS-CoV-2的N基因的crRNA；A crRNA for detecting SARS-CoV-2, characterized in that the crRNA is a crRNA for detecting the N gene of SARS-CoV-2;
    其中，所述crRNA选自如SEQ ID NO:1～4所示的核苷酸序列中的一种或多种。 Wherein, the crRNA is selected from one or more of the nucleotide sequences shown in SEQ ID NO: 1 to 4.
  9. 一种核酸检测的方法，其特征在于，所述方法包括：使待测样品与如权利要求4所述的检测体系发生反应，收集所述反应产生的荧光信号；A method for nucleic acid detection, characterized in that the method includes: reacting the sample to be tested with the detection system as claimed in claim 4, and collecting the fluorescence signal generated by the reaction;
    较佳地，所述反应的时间为5～25min；和/或，所述反应的温度为35～45℃；Preferably, the reaction time is 5 to 25 minutes; and/or the reaction temperature is 35 to 45°C;
    更佳地，所述反应的时间为10～25min，优选地为10～20min，例如为15～20min；和/或，所述反应的温度为35～42℃，优选地为37～42℃，例如为39～42℃。More preferably, the reaction time is 10 to 25 min, preferably 10 to 20 min, such as 15 to 20 min; and/or the reaction temperature is 35 to 42°C, preferably 37 to 42°C, For example, it is 39~42℃.
  10. 如权利要求9所述的方法，其特征在于，所述方法还包括在进行所述反应之前，所述待测样品中的核酸在如权利要求5所述的扩增体系中发生扩增；和/或，所述方法还包括在进行所述反应之前，所述待测样品使用如权利要求6所述的裂解体系进行裂解，以暴露所述待测样品中的核酸；The method of claim 9, further comprising amplifying the nucleic acid in the sample to be tested in the amplification system of claim 5 before performing the reaction; and /Or, the method further includes, before performing the reaction, the sample to be tested is lysed using the cleavage system as claimed in claim 6 to expose the nucleic acid in the sample to be tested;
    较佳地，所述扩增体系与所述检测体系的反应体积比为1:(1～5)；和/或，所述扩增体系预存于发生所述检测反应的封闭容器内；Preferably, the reaction volume ratio of the amplification system and the detection system is 1: (1-5); and/or the amplification system is pre-stored in a closed container where the detection reaction occurs;
    更佳地，所述扩增体系与所述检测体系的反应体积比为1:(2～4)，例如为1:3；和/或，所述检测体系在所述扩增结束后加入所述封闭容器内，所述加入优选通过注射加入；和/或，所述裂解的温度为37℃～95℃，例如为65℃～95℃。More preferably, the reaction volume ratio of the amplification system and the detection system is 1: (2-4), for example, 1:3; and/or, the detection system is added after the amplification is completed. In the closed container, the addition is preferably by injection; and/or the lysis temperature is 37°C to 95°C, for example, 65°C to 95°C.
  11. 一种如权利要求1或2所述的基因编辑系统、如权利要求3所述的核酸、如权利要求4所述的检测体系、如权利要求5所述的扩增体系、如权利要求6所述的裂解体系、如权利要求7所述的试剂盒或者如权利要求8所述的crRNA在制备检测病原核酸的试剂中的应用；A gene editing system as claimed in claim 1 or 2, a nucleic acid as claimed in claim 3, a detection system as claimed in claim 4, an amplification system as claimed in claim 5, as claimed in claim 6 The application of the lysis system, the kit as claimed in claim 7 or the crRNA as claimed in claim 8 in preparing reagents for detecting pathogenic nucleic acids;
    较佳地，所述病原选自病原性真菌、病毒和病原性原核生物；所述病原性原核生物优选包括细菌、支原体和衣原体；Preferably, the pathogen is selected from pathogenic fungi, viruses and pathogenic prokaryotes; the pathogenic prokaryotes preferably include bacteria, mycoplasma and chlamydia;
    更佳地，所述病原为病毒，优选为SARS-CoV-2；所述病原核酸优选为SARS-CoV-2的N基因。 More preferably, the pathogen is a virus, preferably SARS-CoV-2; the pathogenic nucleic acid is preferably the N gene of SARS-CoV-2.
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