WO2022174663A1 - 一种病原体核酸的即时检测系统及方法 - Google Patents

一种病原体核酸的即时检测系统及方法 Download PDF

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WO2022174663A1
WO2022174663A1 PCT/CN2021/138141 CN2021138141W WO2022174663A1 WO 2022174663 A1 WO2022174663 A1 WO 2022174663A1 CN 2021138141 W CN2021138141 W CN 2021138141W WO 2022174663 A1 WO2022174663 A1 WO 2022174663A1
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gene
rpa
sars
sample
reaction
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French (fr)
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赵维
龙飞
杨晓楠
赵国屏
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application belongs to the technical field of nucleic acid detection, and in particular relates to a system and method for instant detection of pathogen nucleic acid.
  • RT-qPCR reverse transcription real-time quantitative polymerase chain reaction
  • RT-qPCR technology first reverse-transcribes viral RNA into cDNA, and then performs polymerase chain reaction.
  • specific fluorescent labeling method which requires the design of a segment complementary to the target DNA.
  • the TaqMan probe has a fluorescent group and a quenching group at both ends of the probe;
  • non-specific fluorescent labeling method which requires the use of fluorescent dyes such as SYBR Green, which can combine with double-stranded DNA to generate fluorescence.
  • the fluorescence generation depends on the combination of the probe and the target DNA. The probe needs to be carefully designed.
  • RT-qPCR and other common nucleic acid detection techniques rely heavily on various sophisticated equipment and are not suitable for self-testing by the general public or use in poor or remote areas with limited resources.
  • the technical problem mainly solved by this application is to provide a system and method for instant detection of pathogen nucleic acid, which can reduce the probability of false positives or false negatives.
  • a technical solution adopted in this application is to provide a real-time detection system for pathogen nucleic acid, including: a lysis buffer for inactivating and lysing the sample to be detected to release RNA; RT premix solution , used to reverse transcribe the RNA in the sample into DNA; RPA premix, used to perform RPA reaction on the sample treated with the RT premix to amplify the DNA in the sample; CRISPR reaction premix , used to carry out a specific cleavage reaction on the sample treated with the RPA premix; colloidal gold test paper, used to develop the color of the sample treated with the CRISPR reaction premix to confirm whether the sample contains the pathogen nucleic acid.
  • the CRISPR reaction premix includes Cas12a protein, crRNA and probe; wherein, the probe includes any one of FB-ssDNA probe, FQ-ssDNA probe; the FB-ssDNA probe is composed of single-stranded DNA and a first group labeled at the end of the single-stranded DNA, the first group includes any one of fluorescein isothiocyanate FITC, 6-carboxyfluorescein FAM, and biotin Biotin, and the The first group can be combined with colloidal gold; the FQ-ssDNA probe is composed of single-stranded DNA and a second group and a second group labeled at the end of the single-stranded DNA, the second group It includes any one of fluorescein isothiocyanate FITC and 6-carboxyfluorescein FAM, the third group includes a black hole quenching group BHQ1, and the third group is used to absorb fluorescence.
  • the probe includes any one of FB-ssDNA probe, FQ
  • the pathogen nucleic acid includes at least one of SARS-CoV2, SARS-CoV, MERS-CoV, and H1N1; wherein, the detection gene of SARS-CoV2 is the SARS-CoV2 N gene, and the corresponding sequence of the crRNA is : 5'-AAUUU CUACU GUUGU AGAU ccaga cauuu ugcuc uca-3'; and/or, the detection gene of SARS-CoV2 is SARS-CoV2 E gene, and the corresponding crRNA sequence is: 5'-AAUUU CUACU GUUGU AGAU caaga cucac guuaa caa-3'; and/or, the detection gene of the SARS-CoV is the SARS-CoV2 N gene, and the corresponding sequence of the crRNA is: 5'-AAUUU CUACU GUUGU AGAU ccaga acuu ugcuc uca-3 '; and/or, the detection gene of the M
  • the CRISPR reaction premix also includes reaction buffer and ultrapure water; wherein, the concentration of crRNA in the mixture formed by the CRISPR reaction premix and a part of the sample after passing through the RPA premix is 0.5uM-1uM, The concentration of the FB-ssDNA probe is 0.5nM-2.0nM, the concentration of the FQ-ssDNA probe is 1nM-10nM, and the concentration of Cas12a protein is 0.1uM-0.5uM.
  • both the RT premix and the RPA premix respectively include RT-RPA primer F and RT-RPA primer R.
  • the pathogen nucleic acid includes one of SARS-CoV2, SARS-CoV, MERS-CoV, and H1N1; wherein, the amplification site of SARS-CoV2 is the SARS-CoV2 N gene, and the corresponding RT-RPA
  • the sequence of primer F is: 5'-CAAGA AATTC AACTC CAGGC AGCAG TAGGG GAAC-3'; the sequence of the RT-RPA primer R is: 5'-CTTTA GTGGC AGTAC GTTTT TGCCG AGGCT TCT-3'; and/or, so
  • the amplification site of SARS-CoV2 is the SARS-CoV2 E gene, and the corresponding sequence of the RT-RPA primer F is: 5'-TACTC ATTCG TTTCG GAAGA GACAG GTACG TT-3'; the RT-RPA primer R
  • the sequence is: 5'-CAGAT TTTTA AACCG AGAGT AAACG TAAAA AGAA-3'.
  • the amplification site of the SARS-CoV is the SARS-CoV N gene
  • the amplification site of the MERS-CoV is the MERS-CoV N gene
  • the amplification site of the H1N1 is the HA1 gene of H1N1 and H1N1 NA1 gene
  • the SARS-CoV N gene, the MERS-CoV gene, the H1N1 HA1 gene and the H1N1 NA1 gene are respectively connected into plasmid pUC18
  • the sequence of the corresponding PCR primer F is: 5'-CCCAGTCACGACGTTGTAAAACG- 3'
  • the sequence of PCR primer R is: 5'-AGCGGATAACAATTTCACACAGG-3'.
  • the RT premix also includes: reaction buffer, RNase inhibitor, dNTP, reverse transcriptase and ultrapure water; wherein, the concentration of the RT-RPA primer F is 0.3uM-0.6uM; the RT - The concentration of RPA primer R is 0.3uM-0.6uM.
  • the RPA premix also includes: reaction buffer, MgOAc and ultrapure water; wherein, the concentration of the RT-RPA primer F is 0.5uM-1.0uM; the concentration of the RT-RPA primer R is 0.5uM -1.0uM.
  • a technical solution adopted in this application is: to provide a method for instant detection of pathogen nucleic acid, the method uses the system mentioned in any of the above embodiments, and the method includes: the sample to be detected is detected. Place in lysis buffer, inactivate and lyse the sample to release RNA; place the sample containing RNA in RT premix to reverse transcribe the RNA into DNA; transcribed the reverse transcribed The sample was placed in the RPA premix to amplify the sample; part of the amplified sample was placed in the CRISPR reaction premix to perform the specific cleavage reaction; the samples after the specific cleavage reaction were visualized with colloidal gold test paper. color.
  • the beneficial effects of the present application are as follows: the three methods of coupling RPA, CRISPR and colloidal gold in this application have huge advantages in nucleic acid detection of pathogens such as new crowns: the high sensitivity of RPA technology can efficiently amplify trace nucleic acids, which solves the problem of routine
  • the problem of low CRISPR sensitivity (false negative) in the CRISPR method crRNA targets the target nucleic acid, and its high specificity eliminates the false positive problem caused by RPA; the CRISPR trans-effect cleavage probe increases the signal by orders of magnitude, which solves the problem of traditional colloidal gold.
  • the problem of low sensitivity (false negative) of test strip technology; the test results presented by colloidal gold test strips are easy to understand and instantly visible.
  • the above system has the advantages of simplicity, sensitivity and specificity at the same time. It does not require instruments and equipment, and is an instant detection system that can quickly obtain results by simple operation at room temperature.
  • 1 is a schematic flowchart of an embodiment of the instant detection method for pathogen nucleic acid of the present application
  • FIG. 2 is a schematic diagram of the SARS-CoV2 virus gene detection site
  • Fig. 3 is the electrophoresis image of SARS-CoV2 virus N gene and E gene RT-RPA product
  • Figure 4 is an electropherogram of CRISPR-Cas12 cis-cut RT-RPA products
  • Fig. 5 is a graph showing the result of colloidal gold detection of CRISPR-Cas12a protein trans-cutting ssDNA probe
  • Fig. 6 is the electrophoresis image of E plasmid RPA product and its cis-cut product by CRISPR-Cas12a protein reaction system;
  • Fig. 7 is the fluorescence detection picture of CRISPR-Cas12a protein reaction system trans-cutting FQ-ssDNA probe
  • Fig. 8 is the schematic diagram of specific detection crRNA sequence and binding site
  • Figure 9 is a schematic diagram of the specific binding effect of SARS-CoV2 N gene crRNA
  • Figure 10 is a fluorescent detection diagram of the SARS-CoV2 N gene crRNA specificity test for trans-cleavage FQ-ssDNA probe
  • Figure 11 is a schematic diagram of crRNA-specific cis-cleavage effect
  • Figure 12 is a graph of fluorescence detection of crRNA-specific trans-cleavage FQ-ssDNA probes.
  • FIG. 1 is a schematic flowchart of an embodiment of the instant detection method for pathogen nucleic acid of the present application. The above method specifically includes:
  • the sample to be detected is placed in a lysis buffer, and the sample is inactivated and lysed to release RNA.
  • the first reverse transcription of RNA in the sample to be detected into DNA can better ensure the stability of the target nucleic acid.
  • the CRISPR reaction master mix can fully complete the cleavage reaction in 30 minutes at 37°C, which is beneficial to improve the efficiency of the detection process.
  • the CRISPR reaction is coupled with the colloidal gold test paper method, which can be detected at any time, and the positive samples and negative samples can be well distinguished, which greatly increases the sensitivity of the colloidal gold test paper method.
  • the above method utilizes a point-of-care detection system for pathogen nucleic acids.
  • the point-of-care detection system for pathogen nucleic acid specifically includes lysis buffer, RT master mix, RPA master mix, CRISPR reaction master mix and colloidal gold test paper.
  • the lysis buffer is used to inactivate and lyse the sample to be detected to release RNA.
  • the formula table of lysis buffer is shown in Table 1.
  • the final concentration of guanidine hydrochloride is 5M-6M, for example, 5M, 5.5M, 6M, etc., which is not limited in this application.
  • the final concentration of Tris-HCl of Tris-HCl is 50mM-150mM, for example, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, etc., which is not made in this application limited.
  • the final concentration of EDTA is 25mM-100mM, for example, 25mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, etc., which is not limited in this application.
  • the final concentration of sodium chloride NaCl is 100mM-200mM, for example, 100mM, 120mM, 140mM, 160mM, 180mM, 200mM, etc., which is not limited in this application.
  • the final concentration of dithiothreitol DTT was 0.5 mM.
  • the lysis buffer also includes polyethylene glycol octyl phenyl ether Triton X-100, and its volume percentage ranges from 0.5% to 5.0%, for example, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, etc., which are not limited in this application.
  • the RT master mix is used to reverse-transcribe RNA in the sample to be detected into DNA.
  • the RT master mix can complete the reverse transcription reaction in 10 minutes at 37°C, which is beneficial to improve the efficiency of the detection process.
  • the RPA premix is used to perform the RPA reaction on the sample after the RT premix, so as to amplify the DNA in the sample.
  • the time for the RPA reaction may be 30 minutes, which is not limited in the present application. This limited degree of pre-amplification facilitates increased sensitivity while reducing the probability of false positives.
  • the CRISPR reaction premix is used to perform a specific cleavage reaction on the sample after passing through the RPA premix.
  • the colloidal gold test paper is used to develop the color of the sample that has undergone the CRISPR reaction premix to confirm whether the sample contains pathogen nucleic acid.
  • the CRISPR reaction master mix specifically includes Cas12a protein, crRNA and probe.
  • the probe includes any one of FB-ssDNA probe and FQ-ssDNA probe.
  • the FB-ssDNA probe consists of single-stranded DNA and a first group labeled at the end of the single-stranded DNA, wherein the first group labeled at the end of the single-stranded DNA Including fluorescein isothiocyanate FITC, 6-carboxyfluorescein FAM, biotin Biotin and other groups that can be applied to colloidal gold.
  • the single-stranded DNA can be in various forms, for example, SEQ ID NO1: GCTAATCG, SEQ ID NO2: GATTA GCGTA CGCAC GTTAC and the like.
  • the complete sequence of the FB-ssDNA probe is: 5'-FITC-GATTA GCGTA CGCAC GTTAC- Biotin-3'.
  • the first group can be replaced with other suitable groups, as long as it can be combined with the colloidal gold quality control line and/or detection line, and the position where the group is connected can also be adjusted.
  • the probe can be determined according to the experimental design of colloidal gold, which is not limited in this application.
  • the FQ-ssDNA probe is composed of single-stranded DNA and the second and third groups labeled at the ends of the single-stranded DNA, wherein the labeling is on the single-stranded DNA.
  • the second group at the end of the strand DNA includes any one of FITC, 6-carboxyfluorescein FAM, the third group includes BHQ1 (Black Hole Quencher1), TAMARA, etc., and the third group The group is used to absorb fluorescence.
  • Single-stranded DNA can be in various forms, e.g., SEQ ID NO1: GCTAATCG, SEQ ID NO2: GATTA GCGTA CGCAC GTTAC, and the like.
  • the complete sequence of the FQ-ssDNA probe is: 5'- FAM-GCTAATCG-BHQ1-3'.
  • the second group and the third group can be replaced with other suitable groups, so that no fluorescence can be detected when the probe is complete, and fluorescence can be detected when the probe is incomplete, and the position where the groups are connected can also be Adjustment.
  • the probe can be determined according to the fluorescence experimental design, which is not limited in this application.
  • the CRISPR system when the CRISPR system does not detect the target nucleic acid in the sample, the system cannot cut the probe, and the complete probe is labeled with two groups at the same time; and when the CRISPR system detects the target nucleic acid in the sample When targeting nucleic acid, the probe will be cleaved into two or more segments by trans-cleaving activity, thereby separating the two groups. In this way, the accuracy and validity of the detection results can be improved.
  • the biotin Biotin on the probe will interact with the colloidal gold Au in the colloidal gold test paper through the streptavidin Streptavidin. Binding, electrophoresed to the first detection band (T band), it was blocked by the anti-fluorescein antibody Anti-F above, and the excess free Au-Streptavidin could interact with the second quality control band (C band). Biotin continued to bind, resulting in color development, and the sample was negative.
  • the fragments labeled with Biotin or FITC will be divided. At this time, only the fragments labeled with Biotin can be The FITC-labeled fragment of fluorescein isothiocyanate that is blocked by anti-fluorescein antibody (Anti-F) binds to colloidal gold in colloidal gold test paper because it is not bound to colloidal gold, and the first detection band (T band) It cannot develop color. Similarly, free Au-Streptavidin can continue to bind to the biotin Biotin on the second quality control band (C band), and then develop color, and the sample is negative. Specifically, the color development time of the colloidal gold test paper is about 5 minutes.
  • the CRISPR reaction is coupled with the colloidal gold test paper method.
  • the CRISPR reaction specifically amplifies the signal of conventional detection and provides the initial detection sample for the colloidal gold test paper method, and the detection signal is read in reverse on the T of the test paper.
  • instant detection can be carried out at any time, which can distinguish positive samples from negative samples well, reduce the probability of false positives and false negatives, greatly increase the sensitivity of the colloidal gold test strip method, and improve the accuracy and flexibility of detection.
  • fluorescence detection when the FQ-ssDNA probe is complete, that is, when the CRISPR system does not detect the target nucleic acid in the sample, the fluorescence emitted by the fluorophore is absorbed by the quenching group, and the fluorescence detection is negative; After the needle is cut, that is, when the target nucleic acid is detected in the sample, the fluorescent group is far away from the quenching group, the emitted fluorescence cannot be absorbed by the quenching group, and the fluorescence detection results in a positive result.
  • the pathogen nucleic acid may comprise at least one of SARS-CoV2, SARS-CoV, MERS-CoV, H1N1.
  • the pathogen nucleic acid may also include at least one of other coronaviruses or other influenza viruses, which is not limited in this application.
  • the detection gene of SARS-CoV2 is SARS-CoV2 N gene, and the sequence of its corresponding crRNA is SEQ ID NO3; and/or, the detection gene of SARS-CoV2 is SARS-CoV2 E gene, and the sequence of its corresponding crRNA is SEQ ID NO3 NO4; and/or, the detection gene of SARS-CoV is SARS-CoV N gene, and its corresponding crRNA sequence is SEQ ID NO5; and/or, the detection gene of MERS-CoV is MERS-CoV N gene, and its corresponding
  • the sequence of crRNA is SEQ ID NO6; and/or, the detection gene of H1N1 is H1N1 HA1, and the sequence of its corresponding crRNA is SEQ ID NO7; and/or, the detection gene of H1N1 is H1N1 NA1, and the sequence of its corresponding crRNA is SEQ ID NO 8.
  • the N gene is the nucleoprotein gene
  • the E gene is the envelope glycoprotein gene
  • the HA1 gene is the hemagglutinin gene (group 1)
  • the NA1 gene is the neuraminidase gene. (Neuraminidase gene, subtype 1).
  • the sequence of the crRNA corresponding to the gene of the positive reference pUC18-LacZ is SEQ ID NO9.
  • the CRISPR reaction master mix also includes reaction buffer and ultrapure water. Specifically, taking the total volume of the CRISPR reaction premix as 100ul as an example, the volume of the reaction buffer is 8.0ul-12.0ul, for example, 8.0ul, 8.5ul, 9.0ul, 9.5ul, 10.0ul, 10.5ul, 11.0ul , 11.5ul, 12.0ul, etc., which are not limited in this application.
  • the original concentration of crRNA in the mixture formed by CRISPR reaction master mix and some samples after RPA master mix is 50uM, and the dosage is 1.0ul-2.0ul, for example, 1.0ul, 1.2ul, 1.4ul, 1.6ul, 1.8ul, 2.0ul ul, etc., which are not limited in this application.
  • the original concentration of the probe is 100nM, and the dosage is 0.5ul-2.0ul, for example, 0.5ul, 0.7ul, 0.9ul, 1.0ul, 1.2ul, 1.4ul, 1.6ul, 1.8ul, 2.0ul, etc.
  • the application is here Not limited.
  • the original concentration of Cas12a protein is 10uM, and the dosage is 1.0ul-5.0ul, for example, 1.0ul, 1.5ul, 2.0ul, 2.5ul, 3.0ul, 3.5ul, 4.0ul, 4.5ul, 5.0ul, etc.
  • the application is here Not limited.
  • the remaining volume was made up with ultrapure water.
  • the total volume of the CRISPR reaction premix and the dosage of each component can be changed, and it only needs to be adjusted according to the concentration ratio of each component, which is not limited in this application.
  • the concentration of crRNA in the mixture formed by the CRISPR reaction premix and the sample after partial RPA premix is 0.5uM-1.0uM, for example, 0.5uM, 0.6uM, 0.7uM, 0.8uM, 0.9uM, 1.0uM, etc., which are not limited in this application.
  • the concentration of FB-ssDNA probe is 0.5nM-2.0nM, for example, 0.5nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM, 1nM, 1.2nM, 1.4nM, 1.6nM, 1.8nM, 2nM, etc., this application It is not limited here.
  • the concentration of FQ-ssDNA probe is 1nM-10nM, for example, 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, etc., which are not limited herein.
  • the concentration of Cas12a protein is 0.1uM-0.5uM, for example, 0.1uM, 0.2uM, 0.3uM, 0.4uM, 0.5uM, etc., which is not limited herein.
  • RT-RPA primer F and RT-RPA primer R are included in RT master mix and RPA master mix, respectively.
  • the pathogen nucleic acid includes at least one of SARS-CoV2, SARS-CoV, MERS-CoV, and H1N1.
  • the pathogen nucleic acid may also include at least one of other coronaviruses or other influenza viruses, which is not limited in this application.
  • the amplification site of SARS-CoV2 is the SARS-CoV2N gene
  • the corresponding sequence of RT-RPA primer F is SEQ ID NO10
  • the sequence of RT-RPA primer R is SEQ ID NO11
  • the amplification site of SARS-CoV2 is SARS-CoV2E gene
  • the sequence of its corresponding RT-RPA primer F is SEQ ID NO12
  • the sequence of RT-RPA primer R is SEQ ID NO13
  • the amplification site of SARS-CoV is SARS-CoV N gene
  • the amplification site of MERS-CoV is MERS-CoV N gene
  • the amplification site of H1N1 is H1N1 HA1 gene and/or or H1N1 NA1 gene
  • RT-RPA primer F corresponding to pUC18-LacZ is SEQ ID NO16
  • sequence of RT-RPA primer R is SEQ ID NO17.
  • the RT master mix further includes reaction buffer, RNase inhibitor, dNTPs, reverse transcriptase, and ultrapure water.
  • the volume of the reaction buffer is 3.2ul-4.8ul, for example, 3.2ul, 3.3ul, 3.4ul, 3.5ul, 3.6ul, 3.7ul, 3.8ul, 3.9ul, 4.0ul, 4.1ul, 4.2ul, 4.3ul, 4.4ul, 4.5ul, 4.6ul, 4.7ul, 4.8ul, etc., which are not limited in this application.
  • the original concentration of the RNase inhibitor is 40U/ul
  • the dosage is 0.3ul-0.5ul, for example, 0.3ul, 0.35ul, 0.4ul, 0.45ul, 0.5ul, etc., which is not limited herein.
  • the original concentration of dNTP is 10mM, and the dosage is 1.5ul-2.0ul, for example, 1.5ul, 1.55ul, 2.0ul, etc., which is not limited in this application.
  • the original concentration of reverse transcriptase is 200U/ul, and the dosage is 0.8ul-1.2ul, for example, 0.8ul, 0.9ul, 1.0ul, 1.1ul, 1.2ul, etc., which is not limited in this application.
  • the remaining volume was made up with ultrapure water.
  • the original concentration of RT-RPA primer F is 25uM, and its dosage is 0.2ul-0.4ul, for example, 0.2ul, 0.25ul, 0.3ul, 0.35ul, 0.4ul, etc., this application It is not limited here.
  • the original concentration of RT-RPA primer R is 25uM, and its dosage is 0.2ul-0.4ul, for example, 0.2ul, 0.25ul, 0.3ul, 0.35ul, 0.4ul, etc., which is not limited in this application.
  • the total volume of the RT premix and the dosage of each component can be changed, and it only needs to be adjusted according to the concentration ratio of each component, which is not limited in this application.
  • the RPA master mix further includes reaction buffer, MgOAc and ultrapure water.
  • the volume of the reaction buffer is 25ul-30ul, for example, 25ul, 26ul, 27ul, 28ul, 29ul, 30ul etc., the application is not limited here.
  • the original concentration of MgOAc is 280nM
  • the dosage is 2.0ul-3.0ul, for example, 2.0ul, 2.25ul, 2.5ul, 2.55ul, 2.7ul, 2.75ul, 2.8ul, 2.85ul, 2.9ul, 2.95ul, 3.0ul, etc., which is not limited in this application.
  • the original concentration of RT-RPA primer F is 25uM, and its dosage is 0.8ul-1.5ul, for example, 0.8ul, 0.9ul, 1.0ul, 1.2ul, 1.4ul, 1.5ul, etc. , which is not limited in this application.
  • the original concentration of RT-RPA primer R is 25uM, and its dosage is 0.8ul-1.5ul, for example, 0.8ul, 0.9ul, 1.0ul, 1.2ul, 1.4ul, 1.5ul, etc., which is not limited in this application.
  • the total volume of the RT premix and the dosage of each component can be changed, and it only needs to be adjusted according to the concentration ratio of each component, which is not limited in this application.
  • the ratio of the volume of RT-RPA product formed by the sample after RPA master mix to the total volume of CRISPR reaction master mix is 0.02-0.10, for example, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05 , 0.06, 0.07, 0.08, 0.09, 0.10, etc., which are not limited.
  • the volume of RT-RPA product in a total volume of 100ul of CRISPR reaction master mix is 2.5ul, which is not limited.
  • the ratio of the total volume of the RT premix to the total volume of the RPA premix is 0.32, and the ratio of the total volume of the RPA premix to the total volume of the CRISPR reaction premix is 0.2.
  • the RT premix is The total volume of the mixed solution is 16ul, the total volume of the RPA premix is 50ul, and the total volume of the CRISPR reaction premix is 100ul, which only needs to meet the above ratios, which are not limited in this application.
  • this application combines three methods of RPA, CRISPR and colloidal gold, which has huge advantages in the nucleic acid detection of pathogens such as the new crown: the high sensitivity of RPA technology can efficiently amplify trace nucleic acids and solve the problem. It solves the problem of low sensitivity (false negative) of conventional CRISPR; crRNA targets the target nucleic acid in the CRISPR method, and its high specificity eliminates the false positive problem caused by RPA; The problem of low sensitivity (false negative) of colloidal gold test paper technology; the test results presented by colloidal gold test paper are simple and easy to understand and can be seen immediately.
  • the above system has the advantages of simplicity, sensitivity and specificity at the same time. It does not require instruments and equipment, and is an instant detection system that can quickly obtain results by simple operation at room temperature.
  • the E gene and the N gene in the SARS-CoV2 virus genome were selected as detection sites.
  • the RT and RPA steps share a set of primers for reverse transcription and amplification of single-stranded RNA samples.
  • Figure 2 is a schematic diagram of the detection site of the SARS-CoV2 virus gene.
  • the RT-RPA amplicon includes the hotspot region of the E gene, and the crRNA binding site partially overlaps with the reverse primer sequence used by many institutions, completely including the site selected by the Chinese Center for Disease Control and Prevention. The binding site partially overlaps with the reverse primer sequence used by the Chinese Center for Disease Control and Prevention.
  • the reverse transcription reaction was performed with the single-stranded RNA sample and its negative control (see Table 4 for details of the reaction system).
  • the reverse transcription reaction was completed by incubating at 37°C for 10 minutes, the reverse transcription product mixture all entered the RPA premix, and the amplification was completed by incubating at 37°C for another 30 minutes.
  • the single-stranded RNA product can be subjected to RT-RPA reaction to obtain a SARS-CoV2 virus amplification molecule of the corresponding size.
  • Figure 3 is the electrophoresis image of the RT-RPA product of the SARS-CoV2 virus N gene and E gene. As shown in Figure 3, A. Lane 1.
  • N gene single-stranded RNA was reacted with RT-RPA to obtain a 219bp product; Lane 2. Negative control did not generate a product after RT-RPA reaction.
  • conventional kits can be used for reverse transcription experiments, and TwistAmp Basic Kit (INTABAS v3.0) of TwistDX TM company is used for RPA.
  • the crRNA fragment consists of a Scaffold fragment at the 5' end and a Spacer fragment at the 3' end: Scaffold is used to bind Cas12a protein, and Spacer is used to bind the target sequence on the double strand of the template DNA.
  • the target sequences are all located within the detection hotspot range described above, as shown in Figure 2.
  • the crRNA sequence was obtained by T7 in vitro transcription, and the principle and method were similar to the preparation of SARS-CoV2 viral RNA described above.
  • the crRNA sequences are shown in Table 2.
  • the sequences of the FQ-ssDNA probe and the FB-ssDNA probe are SEQ ID NO2 and SEQ ID NO1, respectively.
  • the amplified molecule (ie RT-RPA product) of the SARS-CoV2 virus obtained was added to the CRISPR-Cas12 protein reaction system, and after the target site was specifically recognized by the corresponding crRNA, the cis-cleavage effect of the system Can cleave double-stranded DNA near the 3' end of the crRNA binding site.
  • Figure 4 is an electropherogram of CRISPR-Cas12 cis-cut RT-RPA products. As shown in Figure 4, after the RT-RPA products of N gene, E gene and positive control lacZ were recognized by the corresponding crRNA, they were all specifically cut into two short fragments by the CRISPR-Cas12 protein reaction system.
  • C Lane 1. The RT-RPA product of lacZ of 256bp was cut into two fragments of 94bp and 162bp; Lane 2. The reaction solution with the negative product of lacZ RT-RPA added; Lane 3. The N gene of 219bp was not cut by lacZ-crRNA RT-RPA product; Lane 4. RT-RPA product of the lacZ gene.
  • This reaction system adopts New England of Lba Cas12a (Cpf1) kit.
  • the activated CRISPR-Cas12a protein reaction system can also cleave the unrelated FB-ssDNA probe in the system in trans.
  • the principle is as described above, whether the probe is cleaved or not is detected by colloidal gold.
  • Fig. 5 is the result of colloidal gold detection of CRISPR-Cas12a protein trans-cutting FB-ssDNA probe.
  • the system cuts the FB-ssDNA probe in trans, and a C band appears on the colloidal gold, which is a positive result; 2.
  • RT-RPA product There is no N gene in the system. RT-RPA product, the system cannot cut FB-ssDNA probe, T and C bands appear on colloidal gold, which is a negative result; 3. There is non-specific lacZ RT-RPA product in the system, and the system also cannot cut FB-ssDNA probe Needle, negative result. B.1. After the RT-RPA product of the E gene is specifically recognized by its crRNA, the system cuts the FB-ssDNA probe in trans, and a C band appears on the colloidal gold, which is a positive result; 2. There is no RT-RPA of the E gene in the system.
  • the system cuts off the FB-ssDNA probe in trans, and only the C band appears on the colloidal gold (reverse display). If there is no SARS-CoV2 virus target gene in the system, or there is a non-specific nucleic acid gene in the system, the system cannot cut off the FB-ssDNA probe, so two bands of T and C appear on the colloidal gold.
  • Fig. 6 is the electrophoresis image of E plasmid RPA product and its cis-cut product by CRISPR-Cas12a protein reaction system.
  • the E gene stock solution was diluted into a concentration gradient for direct RPA amplification, and electrophoresis was used to detect the success of the RPA reaction and the amount of the product, as shown in A in Figure 6.
  • the RPA product was further subjected to the CRISPR-Cas12a protein reaction, and the cis-cleavage efficiency of the reaction was detected by agarose gel electrophoresis, as shown in Figure 6B.
  • Fig. 7 is the fluorescence detection diagram of FQ-ssDNA probe trans-cut by CRISPR-Cas12a protein reaction system. As shown in Figure 7, if the probe is cleaved in trans to produce green fluorescence and can be clearly distinguished from the negative control, the reaction is successful.
  • the RPA product clearly visible in the electrophoresis of A in Figure 6 was selected for CRISPR-Cas12a protein trans-cleavage reaction. That is, RPA products with a final concentration gradient of E plasmid of 80, 60, 40, 20, and 10 copies/ul were selected as positive samples, and ultrapure water was used as negative control. As shown in Figure 7, the fluorescence curve of the positive samples rose rapidly around 15 minutes after the reaction, and could be completely distinguished from the fluorescence curve of the negative control, while the fluorescence curves of the positive samples were not significantly different.
  • This experiment can specifically detect the N gene and E gene fragments on the SARS-CoV2 virus, and can effectively distinguish the gene fragments that are highly homologous to SARS-CoV and MERS-CoV on the SARS-CoV2 virus.
  • the sequence of the crRNA binding site of the SARS-CoV2 N gene was analyzed.
  • the sequences of SARS-CoV and SARS-CoV2 differed by only 2 bases, and MERS-CoV had a difference of 10 bases.
  • the HA and NA genes of H1N1 No sequence homology to SARS-CoV2.
  • the crRNA binds to the nucleic acid fragment, the cis-cleavage effect will be activated.
  • the crRNA of the SARS-CoV2 N gene can only cut the virus segment corresponding to SARS-CoV2, but cannot cut the N gene segment of other viruses such as SARS-CoV and MERS-CoV, so its specificity can be confirmed.
  • Figure 10 is a fluorescent detection diagram of the SARS-CoV2N gene crRNA specificity test for trans-cut FQ-ssDNA probe. As shown in Figure 10, this experiment confirmed the effect of specific binding of crRNA by detecting whether the CRISPR-Cas12 system can cut FQ-ssDNA probe in trans.
  • the experimental setup is shown in Figure 9.
  • the trans-cleavage effect can be activated, and the FQ-ssDNA probe is cleaved to generate fluorescence. It was found that fluorescence was only produced when the SARS-CoV2 N gene fragment and its crRNA were present in the system.
  • FIG 11 is a schematic diagram of the effect of crRNA-specific cis-cleavage.
  • CRISPR-Cas12 system could cut the template fragment in cis to confirm the specific binding effect of crRNA.
  • lane 1 There is only a 563bp SARS-CoV N gene fragment in the system, and ultrapure water is used to replace crRNA; lane 2.
  • the 563bp SARS-CoV N gene fragment is cut into two short fragments of 336bp and 227bp ; Lane 3.
  • the SARS-CoV2 N gene fragment with a length of 465 bp was not cut.
  • the specificity of the crRNA of the H1N1 NA gene was confirmed to be good.
  • the 563 bp H1N1 NA gene fragment was cut into two short fragments of 326 bp and 237 bp, and the SARS-CoV2 N gene fragment was not cut.
  • Fig. 12 is a graph of fluorescence detection of crRNA-specific trans-cleavage FQ-ssDNA probe.
  • the specific binding effect of crRNA was confirmed by detecting whether the CRISPR-Cas12 system could cut FQ-ssDNA probe in trans.
  • the experimental setup is shown in Figure 11.
  • the trans-cleavage effect can only be activated when the gene fragment specifically binds to crRNA, and the FQ-ssDNA probe is cleaved to generate fluorescence. It was found that only when the fragments in the system corresponded to their crRNAs in pairs, fluorescence was generated. The above four crRNAs cannot specifically bind to the SARS-CoV2N gene fragment, and no false positives will be generated.
  • the coupling of RPA, CRISPR and colloidal gold has great advantages in nucleic acid detection of pathogens such as new crowns: the high sensitivity of RPA technology can efficiently amplify trace nucleic acids, which solves the problem of low sensitivity of conventional CRISPR (false positives).
  • crRNA targets the target nucleic acid, and its high specificity eliminates the false positive problem caused by RPA; the CRISPR trans-effect cleavage probe increases the signal by orders of magnitude, solving the low sensitivity of the traditional colloidal gold test paper technology ( false negatives); the test results presented by colloidal gold test strips are easy to understand and instantly visible.

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Abstract

一种病原体核酸的即时检测系统及方法，所述系统包括：裂解缓冲液，用于对待检测的样品进行灭活裂解，以释放出RNA；RT预混液，用于将样品中的RNA逆转录为DNA；RPA预混液，用于对经RT预混液处理后的样品进行RPA反应，使样品中的DNA扩增；CRISPR反应预混液，用于对经过RPA预混液处理后的样品进行特异性切割反应；胶体金试纸，用于对经过CRISPR反应预混液处理后的样品进行显色，以确认样品中是否包含病原体核酸。通过上述设计方案，将RPA，CRISPR和胶体金三种方法偶联，能够同时拥有简便性、灵敏性和特异性三个优点，无需仪器设备，是室温下简单操作即可快速获取结果的即时检测系统。

Description

一种病原体核酸的即时检测系统及方法 技术领域
本申请属于核酸检测的技术领域，具体涉及一种病原体核酸的即时检测系统及方法。
背景技术
目前所使用的病原体核酸的检测方法通常为逆转录实时定量聚合酶链式反应技术(RT-qPCR)，该方法灵敏度较高、交叉污染较小，是一线医院最常用的检测方法。RT-qPCR技术先将病毒RNA逆转录为cDNA，再进行聚合酶链式反应，它的信号产生一般有两种不同方式：(1)特异性荧光标记法，其需要设计一段和靶标DNA互补的TaqMan探针，探针两端分别带有荧光基团和淬灭基团；(2)非特异性荧光标记法，其需要使用SYBR Green等荧光染料，SYBR Green可以和双链DNA结合产生荧光。第一种方法中，荧光产生依赖于探针和靶标DNA结合，探针需要精心设计，如若模版不完整或者扩增效率低，容易出现假阴性的问题，第二种方法需要对靶标DNA大量扩增，如若样品有DNA污染和非特异的探针结合，则极容易产生假阳性。而且，RT-qPCR以及其他常见的核酸检测技术严重依赖各种精密设备，不适用于普通大众自检或在资源有限的贫困或偏远地区使用。
发明内容
本申请主要解决的技术问题是提供一种病原体核酸的即时检测系统及方法，可以降低出现假阳性或假阴性的概率。
为解决上述技术问题，本申请采用的一个技术方案是：提供一种病原体核酸的即时检测系统，包括：裂解缓冲液，用于对待检测的样品进行灭活裂解，以释放出RNA；RT预混液，用于将所述样品中的RNA逆转录为DNA；RPA预混液，用于对经所述RT预混液处理后的样品进行 RPA反应，使所述样品中的DNA扩增；CRISPR反应预混液，用于对经过所述RPA预混液处理后的样品进行特异性切割反应；胶体金试纸，用于对经过CRISPR反应预混液处理后的样品进行显色，以确认所述样品中是否包含所述病原体核酸。
其中，所述CRISPR反应预混液包括Cas12a蛋白、crRNA以及探针；其中，所述探针包括FB-ssDNA探针、FQ-ssDNA探针中任意一种；所述FB-ssDNA探针由单链DNA以及标记在所述单链DNA端部的第一基团组成，所述第一基团包括异硫氰酸荧光素FITC、6-羧基荧光素FAM、生物素Biotin中任意一种，且所述第一基团能够与胶体金结合；所述FQ-ssDNA探针由单链DNA以及标记在所述单链DNA端部的第二基团和第二基团组成，所述第二基团包括异硫氰酸荧光素FITC、6-羧基荧光素FAM中任意一种，所述第三基团包括黑洞淬灭基团BHQ1，且所述第三基团用于吸收荧光。
其中，所述病原体核酸包括SARS-CoV2、SARS-CoV、MERS-CoV、H1N1中至少一种；其中，所述SARS-CoV2的检测基因为SARS-CoV2 N基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU ccaga cauuu ugcuc uca-3'；和/或，所述SARS-CoV2的检测基因为SARS-CoV2 E基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU caaga cucac guuaa caa-3'；和/或，所述SARS-CoV的检测基因为SARS-CoV2 N基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU ccaga aacuu ugcuc uca-3'；和/或，所述MERS-CoV的检测基因为MERS-CoV N基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU ccaga cucaa gggcu ugu-3'；和/或，所述H1N1的检测基因为H1N1 HA1基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU caguu gcuuc gaaug uua-3'；和/或，所述H1N1的检测基因为H1N1 NA1基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU ggucg cccuc ugauu agu-3'。
其中，所述CRISPR反应预混液还包括反应缓冲液和超纯水；其中，所述CRISPR反应预混液和部分经过所述RPA预混液后的样品所形成混 合物中crRNA的浓度为0.5uM-1uM，所述FB-ssDNA探针的浓度为0.5nM-2.0nM，所述FQ-ssDNA探针的浓度为1nM-10nM，Cas12a蛋白的浓度为0.1uM-0.5uM。
其中，所述RT预混液和所述RPA预混液中均分别包括RT-RPA引物F和RT-RPA引物R。
其中，所述病原体核酸包括SARS-CoV2、SARS-CoV、MERS-CoV、H1N1中一种；其中，所述SARS-CoV2的扩增位点为SARS-CoV2 N基因，对应的所述RT-RPA引物F的序列为：5'-CAAGA AATTC AACTC CAGGC AGCAG TAGGG GAAC-3'；所述RT-RPA引物R的序列为：5'-CTTTA GTGGC AGTAC GTTTT TGCCG AGGCT TCT-3'；和/或，所述SARS-CoV2的扩增位点为SARS-CoV2 E基因，对应的所述RT-RPA引物F的序列为：5'-TACTC ATTCG TTTCG GAAGA GACAG GTACG TT-3'；所述RT-RPA引物R的序列为：5'-CAGAT TTTTA ACACG AGAGT AAACG TAAAA AGAA-3'。
其中，所述SARS-CoV的扩增位点为SARS-CoV N基因，所述MERS-CoV的扩增位点为MERS-CoV N基因，所述H1N1的扩增位点为H1N1的HA1基因和H1N1 NA1基因；所述SARS-CoV N基因、所述MERS-CoV基因、所述H1N1 HA1基因以及所述H1N1 NA1基因分别连入质粒pUC18，对应的PCR引物F的序列为：5'-CCCAGTCACGACGTTGTAAAACG-3'，PCR引物R的序列为：5'-AGCGGATAACAATTTCACACAGG-3'。
其中，所述RT预混液还包括：反应缓冲液、RNA酶抑制剂、dNTP、逆转录酶和超纯水；其中，所述RT-RPA引物F的浓度为0.3uM-0.6uM；所述RT-RPA引物R的浓度为0.3uM-0.6uM。
其中，所述RPA预混液还包括：反应缓冲液、MgOAc和超纯水；其中，所述RT-RPA引物F的浓度为0.5uM-1.0uM；所述RT-RPA引物R的浓度为0.5uM-1.0uM。
为解决上述技术问题，本申请采用的一个技术方案是：提供一种病原体核酸的即时检测方法，所述方法利用上述任一实施例所提及的系统， 所述方法包括：将待检测的样品置于裂解缓冲液中，对所述样品进行灭活裂解，以释放出RNA；将包含RNA的所述样品置于RT预混液中，以将所述RNA逆转录为DNA；将逆转录后的样品置于RPA预混液中，使所述样品扩增；将扩增后的部分样品置于CRISPR反应预混液，以进行特异性切割反应；利用胶体金试纸对特异性切割反应后的样品进行显色。
本申请的有益效果是：本申请中将RPA，CRISPR和胶体金三种方法偶联，在新冠等病原体的核酸检测上有巨大优势：RPA技术的高灵敏度可以高效扩增微量核酸，解决了常规CRISPR灵敏度低(假阴性)的问题；CRISPR方法中crRNA靶向目标核酸，其高特异性消除了RPA带来的假阳性问题；CRISPR反式效应切割探针数量级扩大了信号，解决了传统胶体金试纸技术灵敏度低(假阴性)的问题；胶体金试纸呈现的检测结果简单易懂，即时可见。上述体系同时拥有简便性、灵敏性和特异性三个优点，无需仪器设备，是室温下简单操作即可快速获取结果的即时检测系统。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图，其中：
图1是本申请病原体核酸的即时检测方法一实施方式的流程示意图；
图2是SARS-CoV2病毒基因检测位点示意图；
图3是SARS-CoV2病毒N基因和E基因RT-RPA产物电泳图；
图4是CRISPR-Cas12顺式切割RT-RPA产物电泳图；
图5是CRISPR-Cas12a蛋白反式切割ssDNA探针胶体金检测结果图；
图6是E质粒RPA产物及其被CRISPR-Cas12a蛋白反应体系顺式切割产物电泳图；
图7是CRISPR-Cas12a蛋白反应体系反式切割FQ-ssDNA探针荧光检测图；
图8是特异性检测crRNA序列及结合位点示意图；
图9是SARS-CoV2 N基因crRNA特异性结合效果示意图；
图10是SARS-CoV2 N基因crRNA特异性测试反式切割FQ-ssDNA探针荧光检测图；
图11是crRNA特异性顺式切割效果示意图；
图12是crRNA特异性反式切割FQ-ssDNA探针荧光检测图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本申请的一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本申请保护的范围。
请参阅图1，图1是本申请病原体核酸的即时检测方法一实施方式的流程示意图。上述方法具体包括：
S1：将待检测的样品置于裂解缓冲液中，对样品进行灭活裂解，以释放出RNA。
S2：将包含RNA的样品置于RT预混液中，以将RNA逆转录为DNA。
具体而言，将待检测样品中的RNA先逆转录为DNA更能保证靶标核酸的稳定性。
S3：将逆转录后的样品置于RPA预混液中，使样品扩增。
具体地，通过这种有限程度的预扩增，有利于在提高灵敏度的同时降低出现假阳性的概率。
S4：将扩增后的部分样品置于CRISPR反应预混液，以进行特异性切割反应。
具体而言，CRISPR反应预混液在37℃的情况下30分钟就可以充分 完成切割反应，有利于提高检测过程的效率。
S5：利用胶体金试纸对特异性切割反应后的样品进行显色。
通过上述设计方案，将CRISPR反应与胶体金试纸法进行偶联，可以随时进行即时检测，并且能够很好的分辨出阳性样本和阴性样本，大大增加了胶体金试纸法的灵敏度。
在一个实施方式中，上述方法利用病原体核酸的即时检测系统。病原体核酸的即时检测系统具体包括裂解缓冲液、RT预混液、RPA预混液，CRISPR反应预混液以及胶体金试纸。其中，裂解缓冲液用于对待检测的样品进行灭活裂解，以释放出RNA。
表1裂解缓冲液配方表
成分 终浓度
盐酸胍 5M-6M
三羟甲基氨基甲烷盐酸盐Tris-HCl 50mM-150mM
乙二胺四乙酸EDTA 25mM-100mM
氯化钠NaCl 100mM-200mM
二硫苏糖醇DTT 0.5mM
具体而言，裂解缓冲液的配方表如表1所示。具体地，盐酸胍的终浓度为5M-6M，例如，5M、5.5M、6M等，本申请对此不作限定。三羟甲基氨基甲烷盐酸盐Tris-HCl的终浓度为50mM-150mM，例如，50mM、60mM、70mM、80mM、90mM、100mM、110mM、120mM、130mM、140mM、150mM等，本申请对此不作限定。乙二胺四乙酸EDTA的终浓度为25mM-100mM，例如，25mM、50mM、60mM、70mM、80mM、90mM、100mM等，本申请对此不作限定。氯化钠NaCl的终浓度为100mM-200mM，例如，100mM、120mM、140mM、160mM、180mM、200mM等，本申请对此不作限定。二硫苏糖醇DTT的终浓度为0.5mM。其中，裂解缓冲液还包括聚乙二醇辛基苯基醚Triton X-100，其体积百分数的范围为0.5％-5.0％，例如，0.5％、1.0％、1.5％、2.0％、2.5％、3.0％、3.5％、4.0％、4.5％、5.0％等，本申请对此不作限定。
具体地，在本实施例中，RT预混液用于将待检测样品中的RNA逆转录为DNA。具体而言，RT预混液在37℃的情况下10分钟就可以完成逆转录反应，有利于提高检测过程的效率。
具体地，在本实施例中，RPA预混液用于对经RT预混液后的样品进行RPA反应，以将样品中的DNA进行扩增。具体地，RPA反应的时间可以为30分钟，本申请对此不作限定。通过这种有限程度的预扩增，有利于在提高灵敏度的同时降低出现假阳性的概率。
具体地，在本实施例中，CRISPR反应预混液用于对经过RPA预混液后的样品进行特异性切割反应。
具体地，在本实施例中，胶体金试纸用于对经过CRISPR反应预混液的样品进行显色，以确认样品中是否包含病原体核酸。
通过这种方式，将CRISPR反应与胶体金试纸法进行偶联，能够很好的分辨出阴性样本和阳性样本，大大增加了胶体金试纸法的灵敏度。
表2 CRISPR反应预混液配方表
成分 用量(ul)
crRNA(50uM) 1.0-2.0
探针(100nM) 0.5-2.0
反应缓冲液 8.0-12.0
Cas12a蛋白(10uM) 1.0-5.0
RT-RPA产物 2.5-10
超纯水 补充体系至100
总体积 100
在一个实施方式中，如表2所示，CRISPR反应预混液具体包括Cas12a蛋白、crRNA以及探针。具体而言，探针包括FB-ssDNA探针、FQ-ssDNA探针中任意一种。
具体地，就FB-ssDNA探针而言，FB-ssDNA探针由单链DNA以及标记在单链DNA端部的第一基团组成，其中，标记在单链DNA端部的第一基团包括异硫氰酸荧光素FITC、6-羧基荧光素FAM、生物素Biotin等能适用于胶体金的任意一种基团。其中，单链DNA可以为多种形式， 例如，SEQ ID NO1:GCTAATCG、SEQ ID NO2:GATTA GCGTA CGCAC GTTAC等。例如，当标记在所述单链DNA端部的第一基团为异硫氰酸荧光素FITC和生物素Biotin，FB-ssDNA探针完整的序列为：5'-FITC-GATTA GCGTA CGCAC GTTAC-Biotin-3'。当然，第一基团可以替换为其他适用基团，只要能与胶体金质控线和/或检测线结合即可，基团连接的位置也可以调整。探针可以根据胶体金实验设计来决定，本申请对此不作限定。
具体地，就FQ-ssDNA探针而言，FQ-ssDNA探针由单链DNA以及标记在单链DNA端部的所述第二基团和第三基团组成，其中，标记在所述单链DNA端部的第二基团包括异硫氰酸荧光素FITC、6-羧基荧光素FAM中任意一种，第三基团包括BHQ1(Black Hole Quencher1)、TAMARA等，且所述第三基团用于吸收荧光。单链DNA可以为多种形式，例如，SEQ ID NO1:GCTAATCG、SEQ ID NO2:GATTA GCGTA CGCAC GTTAC等。例如，当标记在所述单链DNA端部的第二基团为6-羧基荧光素FAM，第二基团为黑洞淬灭基团BHQ1，FQ-ssDNA探针完整的序列为：5'-FAM-GCTAATCG-BHQ1-3'。当然，第二基团和第三基团可以替换为其他适用基团，满足探针完整时检出不到荧光，探针不完整时能检出到荧光即可，基团连接的位置也可以调整。探针可以根据荧光实验设计来决定，本申请对此不作限定。
具体地，在本实施例中，当CRISPR体系没有检测到样品中的靶标核酸时，体系不能将探针切割，完整的探针同时标记有两种基团；而当CRISPR体系检测到样品中的靶标核酸时，会行使反式切割活力将探针切割成两段或多段，从而将两种基团分割开来。通过这种方式，可以提高检测结果的准确性和有效性。
具体而言，当FB-ssDNA探针完整时，即CRISPR体系没有检测到样品中的靶标核酸时，探针上的生物素Biotin将通过链霉亲和素Streptavidin与胶体金试纸中的胶体金Au结合，泳动到第一条检测带(T带)，被上面的抗荧光素抗体Anti-F截流显色，而多余游离的Au-Streptavidin能与第二条质控带(C带)上的生物素Biotin继续结合， 进而显色，样品呈阴性。当探针被切割后，即检测到样品中有靶标核酸时，带有生物素Biotin或异硫氰酸荧光素FITC标记的片段会被分割开来，此时，只有生物素Biotin标记的片段能与胶体金试纸中的胶体金结合，被抗荧光素抗体(Anti-F)截流的异硫氰酸荧光素FITC标记的片段由于没有与胶体金结合，此时第一条检测带(T带)并不能显色，同样，游离的Au-Streptavidin能与第二条质控带(C带)上的生物素Biotin继续结合，进而显色，样品呈阴性。具体地，胶体金试纸的显色时间为5分钟左右。
通过这种方式，将CRISPR反应与胶体金试纸法进行偶联，CRISPR反应将常规检测的信号特异性放大后为胶体金试纸法提供起始检测样品，将检测信号反向读取在试纸的T带上，可以随时进行即时检测，能够很好的分辨出阳性样本和阴性样本，降低出现假阳性和假阴性的概率，大大增加胶体金试纸法的灵敏度，提高检测的准确度和灵活性。
就荧光检测具体而言，当FQ-ssDNA探针完整时，即CRISPR体系没有检测到样品中的靶标核酸时，荧光基团发出的荧光被淬灭基团吸收，荧光检测呈阴性结果；当探针被切割后，即检测到样品中有靶标核酸时，荧光基团远离淬灭基团，发出的荧光不能被淬灭基团吸收，荧光检测呈阳性结果。
在一个实施方式中，病原体核酸可以包括SARS-CoV2、SARS-CoV、MERS-CoV、H1N1中的至少一种。当然，在其他实施例中，病原体核酸也可以包括其他冠状病毒或其他流感病毒中的至少一种，本申请在此不作限定。
具体地，在本实施例中，如表3所示。SARS-CoV2的检测基因为SARS-CoV2 N基因，其对应的crRNA的序列为SEQ ID NO3；和/或，SARS-CoV2的检测基因为SARS-CoV2 E基因，其对应的crRNA的序列为SEQ ID NO4；和/或，SARS-CoV的检测基因为SARS-CoV N基因，其对应的crRNA的序列为SEQ ID NO5；和/或，MERS-CoV的检测基因为MERS-CoV N基因，其对应的crRNA的序列为SEQ ID NO6；和/或，H1N1的检测基因为H1N1 HA1，其对应的crRNA的序列为SEQ ID  NO7；和/或，H1N1的检测基因为H1N1 NA1，其对应的crRNA的序列为SEQ ID NO8。其中，N基因为核壳蛋白基因(Nucleoprotein gene)，E基因为包膜糖基因(Envelope glycoprotein gene)，HA1基因为血球凝集素基因(Hemagglutinin gene，group 1)，NA1基因为神经氨酸酶基因(Neuraminidase gene，subtype 1)。
表3 crRNA序列表
Figure PCTCN2021138141-appb-000001
具体地，请继续参阅表3，阳性参照物pUC18-LacZ的基因对应的crRNA的序列为SEQ ID NO9。
在另一个实施方式中，请继续参阅表2，CRISPR反应预混液还包括反应缓冲液和超纯水。具体地，以CRISPR反应预混液的总体积为100ul为例，反应缓冲液的体积为8.0ul-12.0ul，例如，8.0ul、8.5ul、9.0ul、9.5ul、10.0ul、10.5ul、11.0ul、11.5ul、12.0ul等，本申请在此不作限定。CRISPR反应预混液和部分经过RPA预混液后的样品所形成混合物中crRNA的原始浓度为50uM，用量为1.0ul-2.0ul，例如，1.0ul、1.2ul、1.4ul、1.6ul、1.8ul、2.0ul等，本申请在此不作限定。探针的原始浓度为100nM，用量为0.5ul-2.0ul，例如，0.5ul、0.7ul、0.9ul、1.0ul、1.2ul、1.4ul、1.6ul、1.8ul、2.0ul等，本申请在此不作限定。Cas12a 蛋白的原始浓度为10uM，用量为1.0ul-5.0ul，例如，1.0ul、1.5ul、2.0ul、2.5ul、3.0ul、3.5ul、4.0ul、4.5ul、5.0ul等，本申请在此不作限定。其余体积用超纯水补足。当然，在其他实施例中，CRISPR反应预混液的总体积以及其中各成分的用量均可以改变，只需要按照各成分的浓度比例进行调整即可，本申请对此不作限定。具体地，在本实施例中，CRISPR反应预混液和部分经过RPA预混液后的样品所形成混合物中crRNA的浓度为0.5uM-1.0uM，例如，0.5uM、0.6uM、0.7uM、0.8uM、0.9uM、1.0uM等，本申请在此不作限定。FB-ssDNA探针的浓度为0.5nM-2.0nM，例如，0.5nM、0.6nM、0.7nM、0.8nM、0.9nM、1nM、1.2nM、1.4nM、1.6nM、1.8nM、2nM等，本申请在此不作限定。FQ-ssDNA探针的浓度为1nM-10nM，例如，1nM、2nM、3nM、4nM、5nM、6nM、7nM、8nM、9nM、10nM等，本申请在此不作限定。Cas12a蛋白的浓度为0.1uM-0.5uM，例如，0.1uM、0.2uM、0.3uM、0.4uM、0.5uM等，本申请在此不作限定。在又一个实施方式中，如表5和表6所示，RT预混液和RPA预混液中均分别包括RT-RPA引物F和RT-RPA引物R。具体地，在本实施例中，病原体核酸包括SARS-CoV2、SARS-CoV、MERS-CoV、H1N1中的至少一种。当然，在其他实施例中，病原体核酸也可以包括其他冠状病毒或其他流感病毒中的至少一种，本申请在此不作限定。
具体而言，如表4所示，SARS-CoV2的扩增位点为SARS-CoV2N基因，其对应的RT-RPA引物F的序列为SEQ ID NO10，RT-RPA引物R的序列为SEQ ID NO11；和/或，SARS-CoV2的扩增位点为SARS-CoV2E基因，其对应的RT-RPA引物F的序列为SEQ ID NO12，RT-RPA引物R的序列为SEQ ID NO13；和/或，SARS-CoV的扩增位点为SARS-CoV N基因；和/或，MERS-CoV的扩增位点为MERS-CoV N基因；和/或，H1N1的扩增位点为H1N1 HA1基因和/或H1N1 NA1基因，SARS-CoV N基因、MERS-CoV N基因、H1N1 HA1基因和/或H1N1 NA1基因应的PCR引物F的序列为SEQ ID NO14:5’-CCCAG TCACG  ACGTT GTAA AACG-3’，PCR引物R的序列为SEQ ID NO15:5’-AGCGG ATAAC AATTT CACA CAGG-3’。
表4 RT-RPA引物序列表
Figure PCTCN2021138141-appb-000002
具体地，请继续参阅表4，pUC18-LacZ对应的RT-RPA引物F的序列为SEQ ID NO16，RT-RPA引物R的序列为SEQ ID NO17。
在一个实施方式中，如表5所示，RT预混液还包括反应缓冲液、RNA酶抑制剂、dNTP、逆转录酶和超纯水。具体地，以RT预混液的总体积为16ul为例，反应缓冲液的体积为3.2ul-4.8ul，例如，3.2ul、3.3ul、3.4ul、3.5ul、3.6ul、3.7ul、3.8ul、3.9ul、4.0ul、4.1ul、4.2ul、4.3ul、4.4ul、4.5ul、4.6ul、4.7ul、4.8ul等，本申请在此不作限定。
具体地，RNA酶抑制剂的原始浓度为40U/ul，用量为0.3ul-0.5ul，例如，0.3ul、0.35ul、0.4ul、0.45ul、0.5ul等，本申请在此不作限定。将RNA酶抑制剂加入到RT预混液中，浓度为12U/ul-20U/ul，例如，12U/ul、13U/ul、14U/ul、15U/ul、16U/ul、17U/ul、18U/ul、19U/ul、20U/ul等，本申请对此不作限定。dNTP的原始浓度为10mM，用量为1.5ul-2.0ul，例如，1.5ul、1.55ul、2.0ul等，本申请在此不作限定。将dNTP加入到RT预混液中，浓度为1.5x10 -8mM-2.0x10 -8mM，例如，1.5x10 -8mM、1.6x10 -8mM、1.7x10 -8mM、1.8x10 -8mM、1.9x10 -8mM、 2.0x10 -8mM等，在此不作限定。逆转录酶的原始浓度为200U/ul，用量为0.8ul-1.2ul，例如，0.8ul、0.9ul、1.0ul、1.1ul、1.2ul等，本申请在此不作限定。将逆转录酶加入到RT预混液中，浓度为160U/ul-240U/ul，例如，160U/ul、165U/ul、170U/ul、175U/ul、180U/ul、185U/ul、190U/ul、195U/ul、200U/ul、210U/ul、215U/ul、220U/ul、225U/ul、230U/ul、235U/ul、240U/ul等，本申请对此不作限定。其余体积用超纯水补足。
表5 RT预混液配方表
成分 用量(ul)
RT-RPA引物F(25uM) 0.2-0.4
RT-RPA引物R(25uM) 0.2-0.4
反应缓冲液 3.2-4.8
RNA酶抑制剂(40U/ul) 0.3-0.5
dNTP(10mM) 1.5-2.0
逆转录酶(200U/ul) 0.8-1.2
超纯水 补充体系至16
总体积 16
具体而言，如表5所示，RT-RPA引物F的原始浓度为25uM，其用量为0.2ul-0.4ul，例如，0.2ul、0.25ul、0.3ul、0.35ul、0.4ul等，本申请在此不作限定。将RT-RPA引物F加入到RT预混液中，浓度为0.3uM-0.6uM，例如，0.3uM、0.35uM、0.4uM、0.45uM、0.5uM、0.55uM、0.6uM等，本申请在此不作限定。RT-RPA引物R的原始浓度为25uM，其用量为0.2ul-0.4ul，例如，0.2ul、0.25ul、0.3ul、0.35ul、0.4ul等，本申请在此不作限定。将RT-RPA引物R加入到RT预混液中，浓度为0.3uM-0.6uM，例如，0.3uM、0.35uM、0.4uM、0.45uM、0.5uM、0.55uM、0.6uM等，本申请在此不作限定。当然，在其他实施例中，RT预混液的总体积以及其中各成分的用量均可以改变，只需要按照各成分的浓度比例进行调整即可，本申请对此不作限定。
在另一个实施方式中，如表6所示，RPA预混液还包括反应缓冲液、MgOAc和超纯水。具体地，以RPA预混液的总体积为50ul为例，反应 缓冲液的体积为25ul-30ul，例如，25ul、26ul、27ul、28ul、29ul、30ul等，本申请在此不作限定。
表6 RPA预混液配方表
成分 用量(ul)
RT-RPA引物F(25uM) 0.8-1.5
RT-RPA引物R(25uM) 0.8-1.5
反应缓冲液 25-30
MgOAc(280nM) 2.0-3.0
超纯水 补充体系至50
总体积 50
具体地，MgOAc的原始浓度为280nM，用量为2.0ul-3.0ul，例如，2.0ul、2.25ul、2.5ul、2.55ul、2.7ul、2.75ul、2.8ul、2.85ul、2.9ul、2.95ul、3.0ul等，本申请在此不作限定。将MgOAc加入到RPA预混液中，浓度为5.4x10 -7nM-8.4x10 -7nM，例如，5.4x10 -7nM、5.6x10 -7nM、5.8x10 -7nM、6.0x10 -7nM、6.2x10 -7nM、6.4x10 -7nM、6.6x10 -7nM、6.8x10 -7nM、7.0x10 -7nM、7.2x10 -7nM、7.4x10 -7nM、7.6x10 -7nM、7.8x10 -7nM、8.0x10 -7nM、8.2x10 -7nM、8.4x10 -7nM等，本申请在此不作限定。其余体积用超纯水补足。
具体而言，如表6所示，RT-RPA引物F的原始浓度为25uM，其用量为0.8ul-1.5ul，例如，0.8ul、0.9ul、1.0ul、1.2ul、1.4ul、1.5ul等，本申请在此不作限定。将RT-RPA引物F加入RPA预混液中，浓度为0.5uM-1.0uM，例如，0.5uM、0.55uM、0.6uM、0.65uM、0.7uM、0.75uM、0.8uM、0.85uM、0.9uM、0.95uM、1.0uM等，本申请在此不作限定。RT-RPA引物R的原始浓度为25uM，其用量为0.8ul-1.5ul，例如，0.8ul、0.9ul、1.0ul、1.2ul、1.4ul、1.5ul等，本申请在此不作限定。将RT-RPA引物R加入RPA预混液中，浓度为0.5uM-1.0uM，例如，0.5uM、0.55uM、0.6uM、0.65uM、0.7uM、0.75uM、0.8uM、0.85uM、0.9uM、0.95uM、1.0uM等，本申请在此不作限定。当然， 在其他实施例中，RT预混液的总体积以及其中各成分的用量均可以改变，只需要按照各成分的浓度比例进行调整即可，本申请对此不作限定。
具体而言，经过RPA预混液后的样品所形成的RT-RPA产物的体积与CRISPR反应预混液的总体积的比值为0.02-0.10，例如，0.02、0.025、0.03、0.035、0.04、0.045、0.05、0.06、0.07、0.08、0.09、0.10等，对此不作限定。例如，在总体积为100ul的CRISPR反应预混液中RT-RPA产物的体积为2.5ul，对此不作限定。
具体地，在本实施例中，RT预混液的总体积与RPA预混液的总体积的比值为0.32，RPA预混液的总体积与CRISPR反应预混液的总体积的比值为0.2，例如，RT预混液的总体积为16ul，RPA预混液的总体积为50ul，CRISPR反应预混液的总体积为100ul，只需满足以上比例即可，本申请对此不作限定。
总而言之，区别于现有技术的情况，本申请将RPA，CRISPR和胶体金三种方法偶联，在新冠等病原体的核酸检测上有巨大优势：RPA技术的高灵敏度可以高效扩增微量核酸，解决了常规CRISPR灵敏度低(假阴性)的问题；CRISPR方法中crRNA靶向目标核酸，其高特异性消除了RPA带来的假阳性问题；CRISPR反式效应切割探针数量级扩大了信号，解决了传统胶体金试纸技术灵敏度低(假阴性)的问题；胶体金试纸呈现的检测结果简单易懂，即时可见。上述体系同时拥有简便性、灵敏性和特异性三个优点，无需仪器设备，是室温下简单操作即可快速获取结果的即时检测系统。
实施例(以SARS-CoV2病毒为例)：
1、选定SARS-CoV2病毒基因组上特异的检测位点和RT-RPA引物
本实施例选取SARS-CoV2病毒基因组中的E基因和N基因作为检测位点。RT和RPA两个步骤共用一套引物，用于单链RNA样本的逆转录和扩增。
请参阅图2，图2是SARS-CoV2病毒基因检测位点示意图。本实施例中RT-RPA扩增子包含了E基因的热点区，crRNA的结合位点与多家机构采用的反向引物序列部分重合，完全包含了中国疾控中心选取的 位点，crRNA的结合位点与中国疾控中心采用的反向引物序列部分重合。
2、制备SARS-CoV2病毒待测样本
以单链RNA样本及其阴性对照进行逆转录反应(反应体系具体见表4)。逆转录反应由37℃孵育10分钟完成，逆转录产物混合液全部进入RPA预混液，37℃再孵育30分钟完成进行扩增。单链RNA产物经RT-RPA反应可以获得对应大小的SARS-CoV2病毒扩增分子，请参阅图3，图3是SARS-CoV2病毒N基因和E基因RT-RPA产物电泳图。如图3所示，其中A.泳道1.N基因单链RNA经RT-RPA反应后得到219bp的产物；泳道2.阴性对照经RT-RPA反应没有产物生成。B.泳道1.E基因单链RNA经RT-RPA反应后得到192bp的产物；泳道2.阴性对照经RT-RPA反应没有产物生成。C.泳道1.lacZ基因单链RNA经RT-RPA反应后得到256bp的产物；泳道2.阴性对照经RT-RPA反应没有产物生成。其中，逆转录实验可用常规试剂盒，RPA采用TwistDX TM公司的TwistAmp Basic Kit(INTABAS v3.0)。
3、设计合成crRNA和ssDNA探针
在本实施例中，crRNA片段由位于5’端的Scaffold片段和位于3’端的Spacer片段两部分组成：Scaffold用于结合Cas12a蛋白，Spacer用于结合模板DNA双链上的目标序列。目标序列均位于上文所述检测热点范围内，如图2所示。crRNA序列采用T7体外转录获得，原理和方法与上文所述SARS-CoV2病毒RNA制备类似。crRNA序列见表2。FQ-ssDNA探针与FB-ssDNA探针序列分别为SEQ ID NO2和SEQ ID NO1。
4、CRISPR反应预混液顺式切割RT-RPA产物，琼脂糖凝胶电泳检测结果
在本实施例中，将获得的SARS-CoV2病毒的扩增分子(即RT-RPA产物)加入CRISPR-Cas12蛋白反应体系，其中的目标位点被对应crRNA特异识别后，体系的顺式切割效应能在crRNA结合位点3’端附近将双链DNA切开。请参阅图4，图4是CRISPR-Cas12顺式切割RT-RPA产物电泳图。如图4所示，N基因、E基因及阳性对照lacZ的RT-RPA产 物被对应的crRNA识别后，均被CRISPR-Cas12蛋白反应体系特异性切割为两条短片段。将图3获得的阴性产物作为空白对照(模拟临床阴性样本)，将任意其他RT-RPA产物作为非特异对照(模拟临床中非SARS-CoV2病毒的核酸样本)进行同样反应，结果空白对照无可见条带，非特异对照中的核酸不能被反应体系切开。其中，A.泳道1.219bp的N基因RT-RPA产物被切割成100bp和119bp的片段(如箭头所示，两条片段在胶图中难以分开)；泳道2.加入N基因RT-RPA阴性产物的反应液；泳道3.N-crRNA未切开256bp的lacZ的RT-RPA产物；泳道4.N基因RT-RPA产物。B.泳道1.192bp的E基因RT-RPA产物被切割成56bp和136bp的两条片段；泳道2.加入E基因RT-RPA阴性产物的反应液；泳道3.E-crRNA未切开167bp的S基因RT-RPA产物；泳道4.E基因的RT-RPA产物。C.泳道1.256bp的lacZ的RT-RPA产物被切割成94bp和162bp的两条片段；泳道2.加入lacZ RT-RPA阴性产物的反应液；泳道3.lacZ-crRNA未切开219bp的N基因RT-RPA产物；泳道4.lacZ基因的RT-RPA产物。本反应体系采用New England
Figure PCTCN2021138141-appb-000003
的
Figure PCTCN2021138141-appb-000004
Lba Cas12a(Cpf1)试剂盒。
5、CRISPR-Cas12a蛋白反应体系反式切割FB-ssDNA探针，胶体金试纸显色检测结果
在顺式切割RT-RPA产物的同时，激活的CRISPR-Cas12a蛋白反应体系也可以反式切割体系中无关的FB-ssDNA探针。原理如上文所述，探针被切割与否由胶体金检测。如图5所示，图5是CRISPR-Cas12a蛋白反式切割FB-ssDNA探针胶体金检测结果图。其中，A.1.N基因的RT-RPA产物被其crRNA特异识别后，体系反式切割FB-ssDNA探针，在胶体金上出现C带，为阳性结果；2.体系中没有N基因的RT-RPA产物，体系无法切割FB-ssDNA探针，在胶体金上出现T和C带，为阴性结果；3.体系中有非特异的lacZ RT-RPA产物，体系同样无法切割FB-ssDNA探针，为阴性结果。B.1.E基因的RT-RPA产物被其crRNA特异识别后，体系反式切割FB-ssDNA探针，在胶体金上出现C带，为阳性结果；2.体系中没有E基因的RT-RPA产物，体系无法切割 FB-ssDNA探针，在胶体金上出现T和C带，为阴性结果；3.体系中有非特异的S RT-RPA产物，体系同样无法切割FB-ssDNA探针，为阴性结果。C.1.lacZ的RT-RPA产物被其crRNA特异识别后，体系反式切割FB-ssDNA探针，在胶体金上出现C带，为阳性结果；2.体系中没有lacZ的RT-RPA产物，体系无法切割FB-ssDNA探针，在胶体金上出现T和C带，此时为阴性结果；3.体系中有非特异的N基因的RT-RPA产物，体系同样无法切割ssDNA探针，此时为阴性结果。
在CRISPR-Cas12a蛋白反应体系中，如果样本中有SARS-CoV2病毒目的基因，体系反式切断FB-ssDNA探针，在胶体金上只出现C带(反向显示)。如果体系中没有SARS-CoV2病毒目的基因，或者体系中为非特异的核酸基因，体系不能切断FB-ssDNA探针，因此在胶体金上出现T和C两条带。
灵敏度检测
本实验使用含有E基因片段的DNA测试灵敏度。请参阅图6，图6是E质粒RPA产物及其被CRISPR-Cas12a蛋白反应体系顺式切割产物电泳图。将E基因原液稀释成浓度梯度直接进行RPA扩增，电泳检测RPA反应成功与否及产物多少，如图6中A所示。将RPA产物进一步进行CRISPR-Cas12a蛋白反应，通过琼脂糖凝胶电泳检测反应的顺式切割效率，如图6中B所示。本实验采用上文所述FQ-ssDNA探针精确测试体系的最小检测限。本实施例对E基因片段测得的最小检测限为10copy/ul。CRISPR-Cas12a蛋白反应体系在酶标仪中37℃恒温反应1小时，每隔半分钟设置一个检测点。请参阅图7，图7是CRISPR-Cas12a蛋白反应体系反式切割FQ-ssDNA探针荧光检测图。如图7所示，探针被反式切割产生绿色荧光且能与阴性对照明显区分，则表示反应成功。本实验选取在图6中A的电泳中明显可见的RPA产物进行CRISPR-Cas12a蛋白反式切割反应。即是，选取E质粒终浓度梯度为80、60、40、20、10copy/ul的RPA产物为阳性样本，以超纯水为阴性对照。如图7所示，阳性样本的荧光曲线在反应发生15分钟左右时快速上升，能与阴性对照的荧光曲线完全区分开来，而阳性样本之间的荧光曲线没有明显区别。
特异性检测
表7特异性检测片段序列
Figure PCTCN2021138141-appb-000005
本实验能特异检测SARS-CoV2病毒上的N基因和E基因片段，而且，能够有效分辨出SARS-CoV2病毒上与SARS-CoV和MERS-CoV高度同源的基因片段。
本实验以SARS-CoV，SARS-CoV2和MERS-CoV病毒上的N基因检测为例测试了体系的特异性。同时，还选取了症状类似的其他呼吸道疾病如甲型流感病毒H1N1的HA和NA基因片段作为特异性检测的对照样本。基因检测片段序列如图8和表7所示，图8是特异性检测crRNA序列及结合位点示意图。插入于质粒pUC18的Sma I酶切位点。利用pUC18的通用引物M13进行普通PCR扩增即可获得目标片段，无需特定试剂盒。
分析SARS-CoV2 N基因crRNA结合位点的序列，SARS-CoV与SARS-CoV2两者序列只有2个碱基的差异，MERS-CoV与其有10个碱基的差异，H1N1的HA基因和NA基因与SARS-CoV2的序列没有同源性。
在CRISPR-Cas12a蛋白反应体系，如果crRNA与核酸片段结合，则会激活顺式切割效应。SARS-CoV2N基因的crRNA只能切开对应SARS-CoV2的病毒片段，而不能切开其他如SARS-CoV和MERS-CoV病毒的N基因片段，因此，其特异性可以得到证实。
而且，在含有其他四种病毒crRNA的CRISPR/Cas12a蛋白反应体系中分别加入SARS-CoV2的N基因片段，N基因片段也不会被切割，而这些crRNA能特异切割各自对应的片段，请参阅图10，图10是SARS-CoV2N基因crRNA特异性测试反式切割FQ-ssDNA探针荧光检测图。如图10所示，本实验以检测CRISPR-Cas12体系是否能反式切割FQ-ssDNA探针的方法来证实crRNA特异性结合的效果。实验设置如图9所示。当基因片段与crRNA特异结合时才能激活反式切割效应，FQ-ssDNA探针被切割产生荧光。检测发现，只有当体系中同时具有SARS-CoV2 N基因片段及其crRNA时才有荧光产生。
请参阅图11，图11是crRNA特异性顺式切割效果示意图。本实验以检测CRISPR-Cas12体系是否能顺式切割模板片段来证实crRNA特异 性结合的效果。如图11所示，泳道1.体系里只有563bp的SARS-CoV N基因片段，以超纯水替代crRNA；泳道2.563bp的SARS-CoV N基因片段被切割成了336bp和227bp的两条短片段；泳道3.长度为465bp的SARS-CoV2 N基因片段没有被切割。以上结果表明，SARS-CoV N基因的crRNA特异性良好，不会切断SARS-CoV2 N基因。泳道4-6.同理证实MERS-CoV N基因的crRNA的特异性良好，各片段大小与泳道1-3一致；泳道7.体系里只有563bp的H1N1 HA基因片段，以超纯水替代crRNA；泳道8.563bp的H1N1 HA基因片段被切割成了357bp和206bp的两条短片段；泳道9.SARS-CoV2 N基因片段没有被切割。泳道10-12.同理证实H1N1 NA基因的crRNA的特异性良好，563bp的H1N1 NA基因片段被切割成了326bp和237bp的两条短片段，SARS-CoV2 N基因片段没有被切割。
请参阅图12，图12是crRNA特异性反式切割FQ-ssDNA探针荧光检测图。本实验以检测CRISPR-Cas12体系是否能反式切割FQ-ssDNA探针的方法来证实crRNA特异性结合的效果。实验设置如图11所示。如图12所示，当基因片段与crRNA特异结合时才能激活反式切割效应，FQ-ssDNA探针被切割产生荧光。检测发现，只有当体系中片段与其crRNA两两对应时才有荧光产生。上述四种crRNA与SARS-CoV2N基因片段不能特异结合，不会有假阳性产生。
由上述结果可知，将RPA，CRISPR和胶体金三种方法偶联，在新冠等病原体的核酸检测上有巨大优势：RPA技术的高灵敏度可以高效扩增微量核酸，解决了常规CRISPR灵敏度低(假阴性)的问题；CRISPR方法中crRNA靶向目标核酸，其高特异性消除了RPA带来的假阳性问题；CRISPR反式效应切割探针数量级扩大了信号，解决了传统胶体金试纸技术灵敏度低(假阴性)的问题；胶体金试纸呈现的检测结果简单易懂，即时可见。
以上所述仅为本申请的实施例，并非因此限制本申请的专利范围，凡是利用本申请说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其它相关的技术领域，均同理包括在本申请的专利保 护范围内。

Claims (10)

  1. 一种病原体核酸的即时检测系统，其特征在于，包括：
    裂解缓冲液，用于对待检测的样品进行灭活裂解，以释放出RNA；
    RT预混液，用于将所述样品中的RNA逆转录为DNA；
    RPA预混液，用于对经所述RT预混液处理后的样品进行RPA反应，使所述样品中的DNA扩增；
    CRISPR反应预混液，用于对经过所述RPA预混液处理后的样品进行特异性切割反应；
    胶体金试纸，用于对经过CRISPR反应预混液处理后的样品进行显色，以确认所述样品中是否包含所述病原体核酸。
  2. 根据权利要求1所述的系统，其特征在于，所述CRISPR反应预混液包括Cas12a蛋白、crRNA以及探针；
    其中，所述探针包括FB-ssDNA探针、FQ-ssDNA探针中任意一种；所述FB-ssDNA探针由单链DNA以及标记在所述单链DNA端部的第一基团组成，所述第一基团包括异硫氰酸荧光素FITC、6-羧基荧光素FAM、生物素Biotin中任意一种，且所述第一基团能够与胶体金结合；所述FQ-ssDNA探针由单链DNA以及标记在所述单链DNA端部的第二基团和第三基团组成，所述第二基团包括异硫氰酸荧光素FITC、6-羧基荧光素FAM中任意一种，所述第三基团包括黑洞淬灭基团BHQ1，且所述第三基团用于吸收荧光。
  3. 根据权利要求2所述的系统，其特征在于，
    所述病原体核酸包括SARS-CoV2、SARS-CoV、MERS-CoV、H1N1中至少一种；
    其中，所述SARS-CoV2的检测基因为SARS-CoV2 N基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU ccaga cauuu ugcuc uca-3'；
    和/或，所述SARS-CoV2的检测基因为SARS-CoV2 E基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU caaga  cucac guuaa caa-3'；
    和/或，所述SARS-CoV的检测基因为SARS-CoV2 N基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU ccaga aacuu ugcuc uca-3'；
    和/或，所述MERS-CoV的检测基因为MERS-CoV N基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU ccaga cucaa gggcu ugu-3'；
    和/或，所述H1N1的检测基因为H1N1 HA1基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU caguu gcuuc gaaug uua-3'；
    和/或，所述H1N1的检测基因为H1N1 NA1基因，对应的所述crRNA的序列为：5'-AAUUU CUACU GUUGU AGAU ggucg cccuc ugauu agu-3'。
  4. 根据权利要求2所述的系统，其特征在于，
    所述CRISPR反应预混液还包括反应缓冲液和超纯水；
    其中，所述CRISPR反应预混液和部分经过所述RPA预混液后的样品所形成混合物中crRNA的浓度为0.5uM-1uM，所述FB-ssDNA探针的浓度为0.5nM-2.0nM，所述FQ-ssDNA探针的浓度为1nM-10nM，Cas12a蛋白的浓度为0.1uM-0.5uM。
  5. 根据权利要求1所述的系统，其特征在于，
    所述RT预混液和所述RPA预混液中均分别包括RT-RPA引物F和RT-RPA引物R。
  6. 根据权利要求5所述的系统，其特征在于，
    所述病原体核酸包括SARS-CoV2、SARS-CoV、MERS-CoV、H1N1中一种；
    其中，所述SARS-CoV2的扩增位点为SARS-CoV2 N基因，对应的所述RT-RPA引物F的序列为：5'-CAAGA AATTC AACTC CAGGC AGCAG TAGGG GAAC-3'；所述RT-RPA引物R的序列为：5'-CTTTA GTGGC AGTAC GTTTT TGCCG AGGCT TCT-3'；
    和/或，所述SARS-CoV2的扩增位点为SARS-CoV2E基因，对应的所述RT-RPA引物F的序列为：5'-TACTC ATTCG TTTCG GAAGA  GACAG GTACG TT-3'；所述RT-RPA引物R的序列为：5'-CAGAT TTTTA ACACG AGAGT AAACG TAAAA AGAA-3'。
  7. 根据权利要求5所述的系统，其特征在于，
    所述SARS-CoV的扩增位点为SARS-CoV N基因，所述MERS-CoV的扩增位点为MERS-CoV N基因，所述H1N1的扩增位点为H1N1的HA1基因和H1N1 NA1基因；所述SARS-CoV N基因、所述MERS-CoV基因、所述H1N1 HA1基因以及所述H1N1 NA1基因分别连入质粒pUC18，对应的PCR引物F的序列为：5'-CCCAGTCACGACGTTGTAAAACG-3'，PCR引物R的序列为：5'-AGCGGATAACAATTTCACACAGG-3'。
  8. 根据权利要求5所述的系统，其特征在于，所述RT预混液还包括：反应缓冲液、RNA酶抑制剂、dNTP、逆转录酶和超纯水；
    其中，所述RT-RPA引物F的浓度为0.3uM-0.6uM；所述RT-RPA引物R的浓度为0.3uM-0.6uM。
  9. 根据权利要求5所述的系统，其特征在于，所述RPA预混液还包括：反应缓冲液、MgOAc和超纯水；
    其中，所述RT-RPA引物F的浓度为0.5uM-1.0uM；所述RT-RPA引物R的浓度为0.5uM-1.0uM。
  10. 一种病原体核酸的即时检测方法，其特征在于，所述方法利用权利要求1-9中任一项所述的系统，所述方法包括：
    将待检测的样品置于裂解缓冲液中，对所述样品进行灭活裂解，以释放出RNA；
    将包含RNA的所述样品置于RT预混液中，以将所述RNA逆转录为DNA；
    将逆转录后的样品置于RPA预混液中，使所述样品扩增；
    将扩增后的部分样品置于CRISPR反应预混液，以进行特异性切割反应；
    利用胶体金试纸对特异性切割反应后的样品进行显色。
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