CN113061650B - System and method for detecting pathogen nucleic acid in real time - Google Patents
System and method for detecting pathogen nucleic acid in real time Download PDFInfo
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Abstract
The application discloses an instant detection system and method of pathogen nucleic acid, wherein the system comprises: the lysis buffer solution is used for inactivating and lysing the sample to be detected so as to release RNA; RT premix for reverse transcription of RNA in the sample into DNA; the RPA premix is used for carrying out RPA reaction on the sample treated by the RT premix to amplify DNA in the sample; the CRISPR reaction premix is used for carrying out specific cutting reaction on the sample treated by the RPA premix; and the colloidal gold test paper is used for developing the sample treated by the CRISPR reaction premix solution so as to confirm whether pathogen nucleic acid is contained in the sample. Through the design scheme, the three methods of RPA, CRISPR and colloidal gold are coupled, so that the three advantages of simplicity, sensitivity and specificity can be simultaneously possessed, instruments and equipment are not needed, and the real-time detection system capable of rapidly acquiring results can be simply operated at room temperature.
Description
Technical Field
The application belongs to the technical field of nucleic acid detection, and particularly relates to an instant detection system and method for pathogen nucleic acid.
Background
The currently used detection method of pathogen nucleic acid is usually reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR), which has higher sensitivity and less cross contamination, and is the most commonly used detection method in first-line hospitals. RT-qPCR technology, which reverse transcribes viral RNA into cDNA and then carries out the polymerase chain reaction, generally produces signals in two different ways: (1) The specific fluorescent labeling method needs to design a TaqMan probe complementary with target DNA, and two ends of the probe are respectively provided with a fluorescent group and a quenching group; (2) A nonspecific fluorescent labeling method, which requires the use of a fluorescent dye such as SYBR Green, which can bind to double-stranded DNA to generate fluorescence. In the first method, fluorescence generation depends on the combination of probes and target DNA, the probes need to be carefully designed, if templates are incomplete or amplification efficiency is low, false negative problems easily occur, and in the second method, large amounts of target DNA need to be amplified, if samples have DNA pollution and nonspecific probe combination, false positives are easily generated. Moreover, RT-qPCR and other common nucleic acid detection techniques rely heavily on a variety of sophisticated equipment, and are not suitable for common mass self-tests or use in poor or remote areas where resources are limited.
Disclosure of Invention
The application mainly solves the technical problem of providing a system and a method for detecting pathogen nucleic acid in real time, which can reduce the probability of false positive or false negative.
In order to solve the technical problems, the application adopts a technical scheme that: there is provided an on-the-fly detection system for pathogen nucleic acid comprising: the lysis buffer solution is used for inactivating and lysing the sample to be detected so as to release RNA; RT premix for reverse transcription of RNA in the sample into DNA; the RPA premix is used for carrying out RPA reaction on the sample treated by the RT premix so as to amplify DNA in the sample; the CRISPR reaction premix is used for carrying out specific cutting reaction on the sample treated by the RPA premix; and the colloidal gold test paper is used for developing the sample treated by the CRISPR reaction premix solution so as to confirm whether the pathogen nucleic acid is contained in the sample.
Wherein the CRISPR reaction premix comprises Cas12a protein, crRNA, and probes; wherein the probe comprises any one of an FB-ssDNA probe and an FQ-ssDNA probe; the FB-ssDNA probe consists of single-stranded DNA and a first group marked at the end part of the single-stranded DNA, wherein the first group comprises any one of fluorescein isothiocyanate FITC, 6-carboxyfluorescein FAM and Biotin, and the first group can be combined with colloidal gold; the FQ-ssDNA probe consists of single-stranded DNA, a second group and a second group, wherein the second group and the second group are marked at the end part of the single-stranded DNA, the second group comprises any one of fluorescein isothiocyanate FITC and 6-carboxyfluorescein FAM, the third group comprises a black hole quenching group BHQ1, and the third group is used for absorbing fluorescence.
Wherein the pathogen nucleic acid comprises at least one of SARS-CoV2, SARS-CoV, MERS-CoV, H1N 1; wherein the detection gene of SARS-CoV2 is SARS-CoV2N gene, and the corresponding crRNA has the sequence: 5'-AAUUU CUACU GUUGU AGAU ccaga cauuu ugcuc uca-3'; and/or the detection gene of SARS-CoV2 is SARS-CoV2E gene, and the corresponding crRNA has the sequence: 5'-AAUUU CUACU GUUGU AGAU caaga cucac guuaa caa-3'; and/or the detection gene of SARS-CoV is SARS-CoV2N gene, and the corresponding crRNA has the sequence: 5'-AAUUU CUACU GUUGU AGAU ccaga aacuu ugcuc uca-3'; and/or, the detection gene of the MERS-CoV is MERS-CoVN gene, and the corresponding crRNA has the sequence as follows: 5'-AAUUU CUACU GUUGU AGAU ccaga cucaa gggcu ugu-3'; and/or the detection gene of the H1N1 is H1N1HA1 gene, and the corresponding crRNA HAs the sequence: 5'-AAUUU CUACU GUUGU AGAU caguu gcuuc gaaug uua-3'; and/or the detection gene of the H1N1 is the H1N1NA1 gene, and the corresponding crRNA has the sequence as follows: 5'-AAUUU CUACU GUUGU AGAU ggucg cccuc ugauu agu-3'.
Wherein the CRISPR reaction premix further comprises a reaction buffer and ultrapure water; wherein the CRISPR reaction premix and the sample partially passing through the RPA premix form a mixture with a crRNA concentration of 0.5uM-1uM, a FB-ssDNA probe concentration of 0.5nM-2.0nM, a FQ-ssDNA probe concentration of 1nM-10nM, and a Cas12a protein concentration of 0.1uM-0.5uM.
Wherein, the RT-RPA primer F and the RT-RPA primer R are respectively included in the RT premix and the RPA premix.
Wherein the pathogen nucleic acid comprises one of SARS-CoV2, SARS-CoV, MERS-CoV, H1N 1; wherein the amplification site of SARS-CoV2 is SARS-CoV2N gene, and the corresponding RT-RPA primer F has the sequence: 5'-CAAGA AATTC AACTC CAGGC AGCAG TAGGG GAAC-3'; the sequence of the RT-RPA primer R is as follows: 5'-CTTTA GTGGC AGTAC GTTTT TGCCG AGGCT TCT-3'; and/or the amplification site of SARS-CoV2 is SARS-CoV2E gene, and the corresponding sequence of RT-RPA primer F is: 5'-TACTC ATTCG TTTCG GAAGA GACAG GTACG TT-3'; the sequence of the RT-RPA primer R is as follows: 5'-CAGAT TTTTA ACACG AGAGT AAACG TAAAA AGAA-3'.
Wherein the amplification site of SARS-CoV is SARS-CoVN gene, the amplification site of MERS-CoV is MERS-CoV N gene, and the amplification site of H1N1 is HA1 gene of H1N1 and H1N1 NA1 gene; the SARS-CoVN gene, the MERS-CoV gene, the H1N1HA1 gene and the H1N1 NA1 gene are respectively connected into a plasmid pUC18, and the corresponding sequences of PCR primers F are as follows: 5'-CCCAGTCACGACGTTGTAAAACG-3', PCR primer R has the sequence: 5'-AGCGGATAACAATTTCACACAGG-3'.
Wherein, the RT premix still includes: reaction buffer, rnase inhibitor, dNTP, reverse transcriptase and ultrapure water; wherein the concentration of the RT-RPA primer F is 0.3-0.6 uM; the concentration of the RT-RPA primer R is 0.3uM-0.6uM.
Wherein, the RPA premix further comprises: 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.
In order to solve the technical problems, the application adopts a technical scheme that: there is provided a method for the on-the-fly detection of pathogen nucleic acid, the method utilising the system as set out in any one of the embodiments above, the method comprising: placing a sample to be detected in a lysis buffer solution, and inactivating and lysing the sample to release RNA; placing the sample comprising RNA in an RT premix to reverse transcribe the RNA into DNA; placing the sample subjected to reverse transcription in RPA premix liquid to amplify the sample; placing the amplified partial sample in CRISPR reaction premix to perform specific cleavage reaction; and developing the color of the sample subjected to the specific cleavage reaction by using colloidal gold test paper.
The beneficial effects of the application are as follows: the application couples the three methods of RPA, CRISPR and colloidal gold, and has great advantages in the nucleic acid detection of pathogens such as new crown and the like: the high sensitivity of the RPA technology can amplify trace nucleic acid with high efficiency, and solves the problem of low sensitivity (false negative) of the conventional CRISPR; in the CRISPR method, crRNA targets target nucleic acid, and the false positive problem brought by RPA is eliminated with high specificity; the magnitude of the CRISPR trans-effect cutting probe expands the signal, and solves the problem of low sensitivity (false negative) of the traditional colloidal gold test paper technology; the detection result presented by the colloidal gold test paper is simple and easy to understand, and is immediately visible. The system has the advantages of simplicity, sensitivity and specificity, does not need instruments and equipment, and is an instant detection system which can rapidly acquire results by simple operation at room temperature.
Drawings
For a clearer description of the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly introduced below, it being obvious that the drawings in the description below are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art, wherein:
FIG. 1 is a schematic flow chart of an embodiment of a method for detecting pathogen nucleic acid in real time according to the present application;
FIG. 2 is a schematic diagram of SARS-CoV2 virus gene detection site;
FIG. 3 is an electrophoresis diagram of the products of the RT-RPA of the N gene and E gene of SARS-CoV2 virus;
FIG. 4 is a CRISPR-Cas12 cis-cut RT-RPA product electrophoretogram;
FIG. 5 is a graph of the colloidal gold detection results of trans-cleaving ssDNA probes by CRISPR-Cas12a protein;
FIG. 6 is an electrophoretogram of the E plasmid RPA product and its cis-cleaved product by CRISPR-Cas12a protein reaction system;
FIG. 7 is a fluorescent detection graph of a CRISPR-Cas12a protein reaction system trans-cleaved FQ-ssDNA probe;
FIG. 8 is a schematic representation of specific detection of crRNA sequences and binding sites;
FIG. 9 is a schematic diagram showing the effect of SARS-CoV2N gene crRNA specific binding;
FIG. 10 is a fluorescent assay of a SARS-CoV2N gene crRNA specificity test trans-cut FQ-ssDNA probe;
FIG. 11 is a schematic representation of crRNA specific cis-cleavage effects;
FIG. 12 is a fluorescent detection plot of crRNA specific trans-cut FQ-ssDNA probes.
Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Referring to fig. 1, fig. 1 is a flow chart illustrating an embodiment of a method for detecting pathogen nucleic acid in real time according to the present application. The method specifically comprises the following steps:
s1: the sample to be detected is placed in a lysis buffer, and inactivated lysis is performed on the sample to release RNA.
S2: samples containing RNA were placed in RT premix to reverse transcribe RNA into DNA.
Specifically, reverse transcription of RNA in a sample to be detected into DNA can ensure the stability of target nucleic acid.
S3: the reverse transcribed sample was placed in an RPA premix to amplify the sample.
In particular, by such a limited degree of pre-amplification, it is advantageous to reduce the probability of false positives while increasing the sensitivity.
S4: and (3) placing the amplified part of the sample in CRISPR reaction premix to perform a specific cleavage reaction.
Specifically, the CRISPR reaction premix can fully complete the cutting reaction within 30 minutes at 37 ℃, which is beneficial to improving the efficiency of the detection process.
S5: and developing the color of the sample subjected to the specific cleavage reaction by using colloidal gold test paper.
Through the design scheme, the CRISPR reaction is coupled with the colloidal gold test paper method, so that the instant detection can be performed at any time, positive samples and negative samples can be well distinguished, and the sensitivity of the colloidal gold test paper method is greatly improved.
In one embodiment, the above method utilizes an on-the-fly detection system for pathogen nucleic acid. The instant detection system of pathogen nucleic acid specifically comprises lysis buffer solution, RT premix, RPA premix, CRISPR reaction premix and colloidal gold test paper. Wherein, the lysis buffer is used for inactivating and lysing the sample to be detected so as to release RNA.
Table 1 cleavage buffer formulation table
| Composition of the components | Final concentration |
| Guanidine hydrochloride | 5M-6M |
| Tris-HCl (Tris-HCl) hydrochloride | 50mM-150mM |
| Ethylenediamine tetraacetic acid EDTA | 25mM-100mM |
| Sodium chloride NaCl | 100mM-200mM |
| Dithiothreitol DTT | 0.5mM |
Specifically, the formulation table of the lysis buffer is shown in table 1. Specifically, the final concentration of guanidine hydrochloride is 5M-6M, e.g., 5M, 5.5M, 6M, etc., which is not limiting in the present application. The final concentration of Tris-HCl (Tris-HCl) is 50mM-150mM, for example, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, etc., which is not limited in the present application. The final concentration of EDTA is 25mM-100mM, for example, 25mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, etc., and the present application is not limited thereto. The final concentration of NaCl in sodium chloride is 100mM-200mM, for example, 100mM, 120mM, 140mM, 160mM, 180mM, 200mM, etc., and the present application is not limited thereto. The final concentration of dithiothreitol DTT was 0.5mM. Wherein the lysis buffer further comprises polyethylene glycol octyl phenyl ether Triton X-100, the volume percentage of which 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., and the application is not limited thereto.
Specifically, in this example, an RT premix is used to reverse transcribe RNA into DNA in the sample to be tested. Specifically, the RT premix can complete the reverse transcription reaction in 10 minutes at 37 ℃, which is beneficial to improving the efficiency of the detection process.
Specifically, in this example, the RPA premix is used to perform RPA reaction on the sample after RT premix to amplify DNA in the sample. Specifically, the time of RPA reaction may be 30 minutes, which is not limited in the present application. By this limited degree of pre-amplification, it is advantageous to reduce the probability of false positives while increasing sensitivity.
Specifically, in this example, the CRISPR reaction premix is used to perform a specific cleavage reaction on a sample after passing through the RPA premix.
Specifically, in this example, a colloidal gold test paper was used to color the samples that passed through the CRISPR reaction premix to confirm whether pathogen nucleic acids were contained in the samples.
By means of the method, the CRISPR reaction is coupled with the colloidal gold test paper method, negative samples and positive samples can be well distinguished, and the sensitivity of the colloidal gold test paper method is greatly improved.
Table 2 CRISPR reaction premix formula Table
| Composition of the components | Dosage (ul) |
| crRNA(50uM) | 1.0-2.0 |
| Probe (100 nM) | 0.5-2.0 |
| Reaction buffer | 8.0-12.0 |
| Cas12a protein (10 uM) | 1.0-5.0 |
| RT-RPA products | 2.5-10 |
| Ultrapure water | Supplement the system to 100 |
| Total volume of | 100 |
In one embodiment, as shown in table 2, the CRISPR reaction premix specifically includes Cas12a protein, crRNA, and probes. Specifically, the probe includes any one of FB-ssDNA probe and FQ-ssDNA probe.
Specifically, as for the FB-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, wherein the first group labeled at the end of the single-stranded DNA comprises any one group which can be suitably used for colloidal gold such as FITC, 6-carboxyfluorescein FAM, biotin Biotin and the like. Among them, the single-stranded DNA may be in various forms, for example, SEQ ID NO1: GCTAATCG, SEQ ID NO2: GATTA GCGTA CGCAC GTTAC, etc. For example, when the first group labeled at the end of the single-stranded DNA is fluorescein isothiocyanate FITC and Biotin, the complete sequence of the FB-ssDNA probe is: 5'-FITC-GATTA GCGTA CGCAC GTTAC-Biotin-3'. Of course, the first group may be replaced by other suitable groups, so long as the first group can be combined with the colloidal gold quality control line and/or the detection line, and the connection position of the groups can be adjusted. The probe may be determined according to the experimental design of colloidal gold, which is not limited in the present application.
Specifically, as for the FQ-ssDNA probe, the FQ-ssDNA probe is composed of a single-stranded DNA, and the second group and the third group labeled at the end of the single-stranded DNA, wherein the second group labeled at the end of the single-stranded DNA comprises any one of fluorescein isothiocyanate FITC, 6-carboxyfluorescein FAM, the third group comprises BHQ1 (Black Hole Quencher 1), TAMARA, etc., and the third group is used for absorbing fluorescence. Single-stranded DNA may take a variety of forms, e.g., SEQ ID NO1: GCTAATCG, SEQ ID NO2: GATTA GCGTA CGCAC GTTAC, etc. For example, when the second group labeled at the end of the single-stranded DNA is 6-carboxyfluorescein FAM, the second group is black hole quenching group BHQ1, and the complete sequence of FQ-ssDNA probe is: 5'-FAM-GCTAATCG-BHQ1-3'. Of course, the second group and the third group can be replaced by other applicable groups, so that fluorescence can not be detected when the probe is complete, fluorescence can be detected when the probe is incomplete, and the connection position of the groups can be adjusted. The probe may be determined according to a design of a fluorescence experiment, which is not limited in the present application.
Specifically, in this example, when the CRISPR system does not detect the target nucleic acid in the sample, the system cannot cleave the probe, and the complete probe is labeled with both groups; when the CRISPR system detects target nucleic acid in the sample, trans-cleavage activity is exerted to cleave the probe into two or more fragments, so that the two groups are separated. In this way, the accuracy and effectiveness of the detection result can be improved.
Specifically, when the FB-ssDNA probe is complete, i.e. the CRISPR system does not detect target nucleic acid in the sample, biotin on the probe is combined with colloidal gold Au in the colloidal gold test paper through Streptavidin and swims to a first detection zone (T zone), and is cut off and developed by an Anti-fluorescein antibody Anti-F above, and redundant free Au-Streptavidin can be combined with Biotin on a second quality control zone (C zone) continuously, so that color development is carried out, and the sample is negative. When the probe is cut, i.e. the target nucleic acid in the sample is detected, the fragments marked by Biotin or fluorescein isothiocyanate FITC are separated, at this time, only the fragments marked by Biotin can be combined with colloidal gold in the colloidal gold test paper, the fragments marked by fluorescein isothiocyanate FITC intercepted by an Anti-fluorescein antibody (Anti-F) are not combined with the colloidal gold, at this time, the first detection band (T band) cannot develop color, and meanwhile, the free Au-strepitavidine can be combined with Biotin on the second quality control band (C band) continuously, so that color development is carried out, and the sample is negative. Specifically, the color development time of the colloidal gold test paper is about 5 minutes.
By means of the method, the CRISPR reaction is coupled with the colloidal gold test paper method, the CRISPR reaction amplifies the specificity of the conventionally detected signals and provides an initial detection sample for the colloidal gold test paper method, the detection signals are reversely read on the T band of the test paper, instant detection can be carried out at any time, positive samples and negative samples can be well distinguished, the probability of false positive and false negative is reduced, the sensitivity of the colloidal gold test paper method is greatly improved, and the detection accuracy and flexibility are improved.
In the case of fluorescence detection, specifically, when the FQ-ssDNA probe is complete, i.e. the CRISPR system does not detect the target nucleic acid in the sample, the fluorescence emitted by the fluorescent group is absorbed by the quenching group, and the fluorescence detection is a negative result; when the probe is cut, namely, the target nucleic acid in the sample is detected, the fluorescent group is far away from the quenching group, the emitted fluorescence cannot be absorbed by the quenching group, and the fluorescence detection is positive.
In one embodiment, the pathogen nucleic acid may include at least one of SARS-CoV2, SARS-CoV, MERS-CoV, H1N 1. Of course, in other embodiments, the pathogen nucleic acid may also include at least one of other coronaviruses or other influenza viruses, as the application is not limited herein.
Specifically, in the present embodiment, as shown in table 3. The detection gene of SARS-CoV2 is SARS-CoV2N gene, its correspondent crRNA sequence is SEQ ID NO3; and/or the detection gene of SARS-CoV2 is SARS-CoV2E gene, its correspondent crRNA sequence is SEQ ID NO4; and/or the detection gene of SARS-CoV is SARS-CoVN gene, its correspondent crRNA sequence is SEQ ID NO5; and/or the detection gene of the MERS-CoV is MERS-CoVN gene, and the corresponding crRNA sequence is SEQ ID NO6; and/or the detection gene of H1N1 is H1N1HA1, and the corresponding crRNA sequence is SEQ ID NO7; and/or the detection gene of H1N1 is H1N1NA1, and the corresponding crRNA sequence is SEQ ID NO8. Wherein, the N gene is nucleocapsid protein gene (Nucleoproteingene), the E gene is envelope sugar gene (Envelopeglycoprotein gene), the HA1 gene is hemagglutinin gene (Hemagglutiningene, group 1), and the NA1 gene is neuraminidase gene (Neuraminidase gene, subtype 1).
Table 3 crRNA sequence listing
Specifically, please continue to refer to table 3, the sequence of crRNA corresponding to the gene of the positive reference pUC18-LacZ is SEQ ID NO9.
In another embodiment, with continued reference to table 2, the crispr reaction premix further comprises a reaction buffer and ultrapure water. Specifically, taking a total volume of CRISPR reaction premix of 100ul as an example, the volume of reaction buffer is 8.0ul-12.0ul, e.g., 8.0ul, 8.5ul, 9.0ul, 9.5ul, 10.0ul, 10.5ul, 11.0ul, 11.5ul, 12.0ul, etc., the application is not limited herein. The CRISPR reaction premix and the portion of the sample after RPA premix form a mixture having an initial concentration of crRNA of 50. Mu.M in an amount of 1.0ul to 2.0ul, e.g., 1.0ul, 1.2ul, 1.4ul, 1.6ul, 1.8ul, 2.0ul, etc., and the application is not limited thereto. The initial concentration of probe is 100nM and is used in an amount of 0.5ul-2.0ul, e.g., 0.5ul, 0.7ul, 0.9ul, 1.0ul, 1.2ul, 1.4ul, 1.6ul, 1.8ul, 2.0ul, etc., the application is not limited herein. The initial concentration of Cas12a protein is 10uM in an amount of 1.0ul-5.0ul, e.g., 1.0ul, 1.5ul, 2.0ul, 2.5ul, 3.0ul, 3.5ul, 4.0ul, 4.5ul, 5.0ul, etc., the application is not limited herein. The remaining volume was made up with ultrapure water. Of course, in other embodiments, the total volume of the CRISPR reaction premix and the amounts of each component therein may be varied, and only the concentration ratio of each component need be adjusted, which is not limited in the present application. Specifically, in this example, the crRNA concentration in the mixture of CRISPR reaction premix and the sample partially after RPA premix is 0.5uM to 1.0uM, e.g., 0.5uM, 0.6uM, 0.7uM, 0.8uM, 0.9uM, 1.0uM, etc., and the application is not limited thereto. The concentration of the FB-ssDNA probe is 0.5nM to 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., and the present application is not limited thereto. FQ-ssDNA probe concentration is 1nM-10nM, for example, 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, etc., the application is not limited thereto. The concentration of Cas12a protein is 0.1uM-0.5uM, e.g., 0.1uM, 0.2uM, 0.3uM, 0.4uM, 0.5uM, etc., the application is not limited herein. In yet another embodiment, as shown in tables 5 and 6, both RT-RPA primer F and RT-RPA primer R are included in the RT premix and the RPA premix, respectively. In particular, in this embodiment, the pathogen nucleic acid comprises at least one of SARS-CoV2, SARS-CoV, MERS-CoV, H1N 1. Of course, in other embodiments, the pathogen nucleic acid may also include at least one of other coronaviruses or other influenza viruses, as the application is not limited herein.
Specifically, as shown in Table 4, the amplification site of SARS-CoV2 is SARS-CoV2N gene, its corresponding RT-RPA primer F has the sequence of SEQ ID NO10, and the sequence of RT-RPA primer R has the sequence of SEQ ID NO11; and/or the amplification site of SARS-CoV2 is SARS-CoV2E gene, its correspondent RT-RPA primer F sequence is SEQ ID NO12, and the sequence of RT-RPA primer R is SEQ ID NO13; and/or the amplification site of SARS-CoV is SARS-CoV N gene; and/or the amplification site of MERS-CoV is MERS-CoV N gene; and/or the amplification site of H1N1 is H1N1HA1 gene and/or H1N1NA1 gene, the sequence of PCR primer F corresponding to SARS-CoV N gene, MERS-CoV N gene, H1N1HA1 gene and/or H1N1NA1 gene is SEQ ID NO14:5'-CCCAGTCACGACGTTGTAAAACG-3', PCR primer R is SEQ ID NO15:5'-AGCGGATAACAATTTCACACAGG-3'.
TABLE 4RT-RPA primer sequence listing
Specifically, with continued reference to Table 4, the sequence of the RT-RPA primer F corresponding to pUC18-LacZ is SEQ ID NO16, and the sequence of the RT-RPA primer R is SEQ ID NO17.
In one embodiment, as shown in table 5, the RT premix further comprises a reaction buffer, an rnase inhibitor, dntps, reverse transcriptase, and ultrapure water. Specifically, the volume of the reaction buffer is 3.2ul to 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., by taking the total volume of the RT premix as an example, the application is not limited herein.
Specifically, the RNase inhibitor is used in an amount of 0.3ul to 0.5ul, for example, 0.3ul, 0.35ul, 0.4ul, 0.45ul, 0.5ul, etc., at an original concentration of 40U/ul, and the present application is not limited thereto. RNase inhibitors are added to the RT premix at a concentration of 12U/ul to 20U/ul, for example, 12U/ul, 13U/ul, 14U/ul, 15U/ul, 16U/ul, 17U/ul, 18U/ul, 19U/ul, 20U/ul, etc., as the present application is not limited thereto. The initial concentration of dNTPs is 10mM and the amount is 1.5ul-2.0ul, for example, 1.5ul, 1.55ul, 2.0ul, etc., and the present application is not limited thereto. dNTPs are added to the RT premix at a concentration of 1.5X10 -8mM-2.0x10-8 mM, e.g., ,1.5x10-8mM、1.6x10-8mM、1.7x10-8mM、1.8x10-8mM、1.9x10-8mM、2.0x10-8mM, etc., without limitation. The original concentration of reverse transcriptase is 200U/ul and the amount is 0.8ul to 1.2ul, for example, 0.8ul, 0.9ul, 1.0ul, 1.1ul, 1.2ul, etc., and the present application is not limited thereto. Reverse transcriptase is added to RT premix at a concentration of 160U/ul to 240U/ul, e.g., ,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, etc., as not limiting in this regard. The remaining volume was made up with ultrapure water.
TABLE 5RT premix formulation table
| Composition of the components | Dosage (ul) |
| RT-RPA primer F (25 uM) | 0.2-0.4 |
| RT-RPA primer R (25 uM) | 0.2-0.4 |
| Reaction buffer | 3.2-4.8 |
| RNase inhibitor (40U/ul) | 0.3-0.5 |
| dNTP(10mM) | 1.5-2.0 |
| Reverse transcriptase (200U/ul) | 0.8-1.2 |
| Ultrapure water | Make up system to 16 |
| Total volume of | 16 |
Specifically, as shown in Table 5, the original concentration of RT-RPA primer F is 25. Mu.M, and the amount thereof is 0.2. Mu.l to 0.4. Mu.l, for example, 0.2. Mu.l, 0.25. Mu.l, 0.3. Mu.l, 0.35. Mu.l, 0.4. Mu.l, etc., and the present application is not limited thereto. The RT-RPA primer F is added to the RT premix at a concentration of 0.3uM to 0.6uM, for example, 0.3uM, 0.35uM, 0.4uM, 0.45uM, 0.5uM, 0.55uM, 0.6uM, etc., and the application is not limited thereto. The original concentration of RT-RPA primer R is 25. Mu.M, and the amount thereof is 0.2. Mu.l to 0.4. Mu.l, for example, 0.2. Mu.l, 0.25. Mu.l, 0.3. Mu.l, 0.35. Mu.l, 0.4. Mu.l, etc., and the present application is not limited thereto. The RT-RPA primer R is added to the RT premix at a concentration of 0.3uM to 0.6uM, for example, 0.3uM, 0.35uM, 0.4uM, 0.45uM, 0.5uM, 0.55uM, 0.6uM, etc., and the present application is not limited thereto. Of course, in other embodiments, the total volume of the RT premix and the amounts of the components therein may be varied, and only the concentration ratio of the components may be adjusted, which is not limited by the present application.
In another embodiment, as shown in Table 6, the RPA premix further includes a reaction buffer, mgOAc, and ultrapure water. Specifically, taking the total volume of the RPA premix as 50ul as an example, the volume of the reaction buffer is 25ul-30ul, for example, 25ul, 26ul, 27ul, 28ul, 29ul, 30ul, etc., the present application is not limited thereto.
TABLE 6RPA premix formulation Table
| Composition of the components | Dosage (ul) |
| RT-RPA primer F (25 uM) | 0.8-1.5 |
| RT-RPA primer R (25 uM) | 0.8-1.5 |
| Reaction buffer | 25-30 |
| MgOAc(280nM) | 2.0-3.0 |
| Ultrapure water | Replenishing the system to 50 |
| Total volume of | 50 |
Specifically, the initial concentration of MgOAc is 280nM and is used in an amount of 2.0ul-3.0ul, e.g., 2.0ul, 2.25ul, 2.5ul, 2.55ul, 2.7ul, 2.75ul, 2.8ul, 2.85ul, 2.9ul, 2.95ul, 3.0ul, etc., the application is not limited thereto. MgOAc is added to the RPA premix at a concentration of 5.4x10 -7nM-8.4x10-7 nM, e.g., ,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, etc., the application is not limited herein. The remaining volume was made up with ultrapure water.
Specifically, as shown in Table 6, the original concentration of RT-RPA primer F is 25. Mu.M, and the amount thereof is 0.8. Mu.l to 1.5. Mu.l, for example, 0.8. Mu.l, 0.9. Mu.l, 1.0. Mu.l, 1.2. Mu.l, 1.4. Mu.l, 1.5. Mu.l, etc., and the present application is not limited thereto. RT-RPA primer F is added to the RPA premix at a concentration of 0.5uM-1.0uM, for example, 0.5uM, 0.55uM, 0.6uM, 0.65uM, 0.7uM, 0.75uM, 0.8uM, 0.85uM, 0.9uM, 0.95uM, 1.0uM, etc., and the present application is not limited thereto. The original concentration of RT-RPA primer R is 25. Mu.M, and the amount thereof is 0.8ul-1.5ul, for example, 0.8ul, 0.9ul, 1.0ul, 1.2ul, 1.4ul, 1.5ul, etc., and the present application is not limited thereto. The RT-RPA primer R is added to the RPA premix at a concentration of 0.5uM to 1.0uM, for example, 0.5uM, 0.55uM, 0.6uM, 0.65uM, 0.7uM, 0.75uM, 0.8uM, 0.85uM, 0.9uM, 0.95uM, 1.0uM, etc., and the present application is not limited thereto. Of course, in other embodiments, the total volume of the RT premix and the amounts of the components therein may be varied, and only the concentration ratio of the components may be adjusted, which is not limited by the present application.
Specifically, the ratio of the volume of the RT-RPA product formed by the sample after passing through the RPA premix to the total volume of the CRISPR reaction premix 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., without limitation. For example, the volume of RT-RPA product in the CRISPR reaction premix solution is 2.5ul in a total volume of 100ul, which is not limited.
Specifically, in this embodiment, the ratio of the total volume of the RT premix to the total volume of the RPA premix is 0.32, the ratio of the total volume of the RPA premix to the total volume of the CRISPR reaction premix is 0.2, for example, 16ul of the total volume of the RT premix, 50ul of the total volume of the RPA premix, and 100ul of the total volume of the CRISPR reaction premix, which is not limited in this application.
In summary, unlike the prior art, the present application couples three methods, RPA, CRISPR and colloidal gold, with great advantages in nucleic acid detection of novel crown pathogens: the high sensitivity of the RPA technology can amplify trace nucleic acid with high efficiency, and solves the problem of low sensitivity (false negative) of the conventional CRISPR; in the CRISPR method, crRNA targets target nucleic acid, and the false positive problem brought by RPA is eliminated with high specificity; the magnitude of the CRISPR trans-effect cutting probe expands the signal, and solves the problem of low sensitivity (false negative) of the traditional colloidal gold test paper technology; the detection result presented by the colloidal gold test paper is simple and easy to understand, and is immediately visible. The system has the advantages of simplicity, sensitivity and specificity, does not need instruments and equipment, and is an instant detection system which can rapidly acquire results by simple operation at room temperature.
Examples (for SARS-CoV2 virus):
1. Selection of specific detection sites and RT-RPA primers on SARS-CoV2 Virus genome
The E gene and N gene in SARS-CoV2 virus genome are selected as detection sites in this example. The RT and RPA steps share a set of primers for reverse transcription and amplification of single stranded RNA samples.
Referring to FIG. 2, FIG. 2 is a schematic diagram showing the detection sites of SARS-CoV2 virus gene. In this embodiment, the RT-RPA amplicon includes the hot spot region of the E gene, the binding site of crRNA partially coincides with the reverse primer sequence adopted by multiple institutions, and completely includes the site selected by the Chinese disease control center, and the binding site of crRNA partially coincides with the reverse primer sequence adopted by the Chinese disease control center.
2. Preparation of SARS-CoV2 Virus test sample
Reverse transcription was performed with single stranded RNA samples and their negative controls (reaction systems are specifically shown in Table 4). The reverse transcription reaction was completed by incubation at 37℃for 10 minutes, and the whole of the reverse transcription product mixture was put into RPA premix, and further incubation at 37℃for 30 minutes was completed for amplification. The amplified molecules of SARS-CoV2 virus with corresponding size can be obtained by RT-RPA reaction of single-stranded RNA product, refer to FIG. 3, and FIG. 3 is an electrophoresis chart of SARS-CoV2 virus N gene and E gene RT-RPA products. As shown in FIG. 3, wherein A. Lane 1.N gene single-stranded RNA is subjected to RT-RPA reaction to obtain 219bp product; lane 2. Negative control no product was produced by RT-RPA reaction. B. Lane 1.E gene single-stranded RNA reacted by RT-RPA to obtain 192bp product; lane 2. Negative control no product was produced by RT-RPA reaction. C. Lane 1. The single stranded RNA of the lacZ gene gave a 256bp product after RT-RPA reaction; lane 2. Negative control no product was produced by RT-RPA reaction. Among them, conventional kits can be used for the reverse transcription assay, and TwistAmp Basic Kit (INTABAS v 3.0) from TwistDX TM company is used for RPA.
3. Design and Synthesis of crRNA and ssDNA probes
In this example, 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 to the Cas12a protein and Spacer is used to bind to the target sequence on the template DNA duplex. The target sequences were all located within the detection hotspots described above, as shown in FIG. 2. The crRNA sequence was obtained by T7 in vitro transcription, and the principle and method were similar to those described above for SARS-CoV2 viral RNA preparation. The crRNA sequences are shown in Table 2. The FQ-ssDNA probe and the FB-ssDNA probe have the sequences of SEQ ID NO2 and SEQ ID NO1 respectively.
4. CRISPR reaction premix cis-cuts RT-RPA product, agarose gel electrophoresis detection result
In this example, the amplified molecules of SARS-CoV2 virus (i.e., RT-RPA products) obtained are added to a CRISPR-Cas12 protein reaction system, where after the target site is specifically recognized by the corresponding crRNA, the cis-cleavage effect of the system is capable of cleaving double-stranded DNA near the 3' end of the crRNA binding site. Referring to fig. 4, fig. 4 is a CRISPR-Cas12 cis-cut RT-RPA product electrophoretogram. As shown in fig. 4, after the N gene, E gene and RT-RPA product of positive control lacZ are recognized by the corresponding crrnas, they are specifically cleaved into two short fragments by CRISPR-Cas12 protein reaction system. The same reaction was performed using the negative product obtained in FIG. 3 as a blank (a simulated clinical negative sample) and any other RT-RPA product as a non-specific control (a simulated clinical nucleic acid sample of non-SARS-CoV 2 virus), so that the blank had no visible band and the nucleic acid in the non-specific control could not be cut by the reaction system. Wherein, lane 1.219bp N gene RT-RPA product is cut into fragments of 100bp and 119bp (as shown by the arrow, the two fragments are difficult to separate in the gel diagram); lane 2, reaction solution containing negative product of RT-RPA of N gene; lane 3.N-crRNA does not cleave the RT-RPA product of 256bp lacZ; lane 4. RT-RPA products of the N gene. B. Lane 1.192bp E gene RT-RPA was cut into two fragments of 56bp and 136 bp; lane 2, reaction solution containing E gene RT-RPA negative product; lanes 3.E-crRNA did not cut 167bp of the S gene RT-RPA product; lane 4.E RT-RPA product of the gene. C. The RT-RPA product of the 1.256bp lacZ in lane was cut into two fragments of 94bp and 162 bp; lane 2. Reaction solution containing lacZRT-RPA negative product; lane 3.LacZ-crRNA does not cleave 219bp of the N gene RT-RPA product; lane 4. RT-RPA product of lacZ gene. The reaction system adopts NEW ENGLANDA kind of electronic deviceAn Lba Cas12a (Cpf 1) kit.
5. Trans-cutting FB-ssDNA probe of CRISPR-Cas12a protein reaction system, and developing detection result by colloidal gold test paper
At the same time as the cis-cleavage of the RT-RPA product, 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, and whether the probe is cleaved or not is detected by colloidal gold. As shown in FIG. 5, FIG. 5 is a graph of the colloidal gold detection result of the trans-cleaving of the CRISPR-Cas12a protein by the FB-ssDNA probe. After the RT-RPA product of the A.1.N gene is specifically identified by crRNA, the system trans-cuts the FB-ssDNA probe, and a C band appears on colloidal gold, which is a positive result; 2. the system has no RT-RPA product of the N gene, the system can not cut FB-ssDNA probes, T and C bands appear on colloidal gold, and a negative result is obtained; 3. the system has nonspecific lacZRT-RPA product, and the system can not cut FB-ssDNA probe, thus being a negative result. After the RT-RPA product of the E gene is specifically identified by crRNA, the system trans-cuts the FB-ssDNA probe, and a C band appears on colloidal gold, which is a positive result; 2. the system has no RT-RPA product of the E gene, the system can not cut FB-ssDNA probes, T and C bands appear on colloidal gold, and a negative result is obtained; 3. the system has a nonspecific S RT-RPA product, and the system can not cut the FB-ssDNA probe, so that a negative result is obtained. After the RT-RPA product of lacZ is specifically identified by crRNA, the system trans-cleaves FB-ssDNA probe, and C band appears on colloidal gold, which is a positive result; 2. the system has no RT-RPA product of lacZ, the system can not cut FB-ssDNA probe, T and C bands appear on colloidal gold, and the result is a negative result; 3. the system has RT-RPA products of non-specific N genes, and the system can not cut ssDNA probes, so that the system is a negative result.
In the CRISPR-Cas12a protein reaction system, if SARS-CoV2 virus target gene exists in the sample, the system cuts off the FB-ssDNA probe in trans, and only the C band appears on the colloidal gold (reverse display). If the system does not have SARS-CoV2 virus target gene or is nonspecific nucleic acid gene, the system can not cut off FB-ssDNA probe, so that T and C bands appear on colloidal gold.
Sensitivity detection
The sensitivity was tested using DNA containing the E gene fragment. Referring to fig. 6, fig. 6 is an electrophoretogram of the E plasmid RPA product and its cis-cleaved product by CRISPR-Cas12a protein reaction system. E gene stock solution is diluted into concentration gradient to directly carry out RPA amplification, and electrophoresis is used for detecting whether the RPA reaction is successful or not and how much product is produced, as shown in A in figure 6. The RPA products were further subjected to CRISPR-Cas12a protein reaction, and the cis-cleavage efficiency of the reaction was detected by agarose gel electrophoresis, as shown in B in fig. 6. The experiment uses the minimum limit of detection of the FQ-ssDNA probe accurate test system described above. The minimum detection limit for the E gene fragment measured in this example was 10copy/ul. The CRISPR-Cas12a protein reaction system reacts for 1 hour at a constant temperature of 37 ℃ in an enzyme label instrument, and a detection point is arranged every half minute. Referring to fig. 7, fig. 7 is a fluorescent detection diagram of trans-cleaved FQ-ssDNA probes of a CRISPR-Cas12a protein reaction system. As shown in FIG. 7, the probe was trans-cleaved to generate green fluorescence and was clearly distinguishable from the negative control, indicating successful reaction. This experiment selects RPA products that are clearly visible in the electrophoresis of a in fig. 6 for CRISPR-Cas12a protein trans-cleavage reactions. Namely, RPA products with final concentration gradients of 80, 60, 40, 20, 10copy/ul of E plasmid were selected as positive samples, and ultrapure water was used as negative control. As shown in fig. 7, the fluorescence curve of the positive sample rapidly rises when the reaction occurs for about 15 minutes, and can be completely distinguished from the fluorescence curve of the negative control, while the fluorescence curves between the positive samples are not significantly different.
Specific detection
TABLE 7 specific detection of fragment sequences
The experiment can specifically detect N gene and E gene fragments on SARS-CoV2 virus, and can effectively distinguish the gene fragments on SARS-CoV2 virus, which are highly homologous with SARS-CoV and MERS-CoV.
The specificity of the system was tested in this experiment using the detection of the N gene on SARS-CoV, SARS-CoV2 and MERS-CoV viruses as an example. Meanwhile, HA and NA gene segments of other respiratory diseases with similar symptoms, such as influenza A virus H1N1, are also selected as control samples for specific detection. The sequences of the gene detection fragments are shown in FIG. 8 and Table 7, and FIG. 8 is a schematic diagram of specific detection of crRNA sequences and binding sites. Inserted into the SmaI cleavage site of the plasmid pUC 18. The target fragment can be obtained by common PCR amplification using the universal primer M13 of pUC18 without a specific kit.
Analyzing the sequence of the crRNA binding site of SARS-CoV 2N gene, the difference between the sequences of SARS-CoV and SARS-CoV2 is only 2 bases, the difference between MERS-CoV and MERS-CoV is 10 bases, and the HA gene and NA gene of H1N1 have no homology with the sequence of SARS-CoV 2.
In the CRISPR-Cas12a protein reaction system, if crRNA is bound to a nucleic acid fragment, the cis-cleavage effect is activated. The crRNA of SARS-CoV2N gene can only cleave the viral fragment corresponding to SARS-CoV2, but cannot cleave other N gene fragments such as SARS-CoV and MERS-CoV viruses, and thus its specificity can be confirmed.
Furthermore, the N gene fragments of SARS-CoV2 are added into CRISPR/Cas12a protein reaction system containing other four kinds of viral crRNAs, the N gene fragments are not cut, and the crRNAs can specifically cut the corresponding fragments, please refer to FIG. 10, FIG. 10 is a fluorescence detection diagram of a trans-cut FQ-ssDNA probe for SARS-CoV2N gene crRNA specificity test. As shown in fig. 10, the present experiment demonstrates the effect of crRNA specific binding in a method to detect whether the CRISPR-Cas12 system can trans-cleave the FQ-ssDNA probe. The experimental setup is shown in fig. 9. The trans-cleavage effect is activated when the gene fragment specifically binds to crRNA and the FQ-ssDNA probe is cleaved to generate fluorescence. It was found that fluorescence was only generated when SARS-CoV2N gene fragment and crRNA thereof were simultaneously present in the system.
Referring to FIG. 11, FIG. 11 is a schematic diagram showing crRNA-specific cis-cleavage effect. This experiment demonstrates the effect of crRNA specific binding by detecting whether the CRISPR-Cas12 system is able to cleave the template fragment in cis. As shown in FIG. 11, lane 1. Only the 563bp SARS-CoV N gene fragment was present in the system, and the crRNA was replaced with ultrapure water; the SARS-CoV N gene fragment of 2.563bp in lane is cut into two short fragments of 336bp and 227 bp; lane 3. SARS-CoV2N gene fragment of 465bp in length was not cleaved. The above results indicate that the crRNA of SARS-CoV N gene has good specificity and does not cleave the SARS-CoV2N gene. Lanes 4-6. Similarly, the crRNA of the MERS-CoV N gene was confirmed to have good specificity, and the sizes of the fragments were consistent with lanes 1-3; lane 7. Only the 563bp H1N1 HA gene fragment was used to replace crRNA with ultrapure water; the H1N1 HA gene fragment of 8.563bp was cut into two short fragments of 357bp and 206bp in lane; lane 9.SARS-CoV2N gene fragment was not cleaved. Lanes 10-12. Similarly, it was confirmed that the crRNA of the H1N1 NA gene had good specificity, the 563bp H1N1 NA gene fragment was cut into two short fragments of 326bp and 237bp, and the SARS-CoV2N gene fragment was not cut.
Referring to FIG. 12, FIG. 12 is a fluorescent detection graph of crRNA-specific trans-cut FQ-ssDNA probes. This experiment demonstrates the effect of crRNA specific binding in a method to detect whether the CRISPR-Cas12 system can trans-cleave the FQ-ssDNA probe. The experimental setup is shown in fig. 11. As shown in FIG. 12, the trans-cleavage effect is activated when the gene fragment specifically binds to crRNA and the FQ-ssDNA probe is cleaved to generate fluorescence. The assay found that fluorescence was only generated when fragments in the system corresponded to their crrnas in pairs. The four crRNAs can not be specifically combined with SARS-CoV 2N gene fragment, and false positive can not be generated.
From the above results, it can be seen that coupling the three methods of RPA, CRISPR and colloidal gold has great advantages in nucleic acid detection of pathogens such as new crown: the high sensitivity of the RPA technology can amplify trace nucleic acid with high efficiency, and solves the problem of low sensitivity (false negative) of the conventional CRISPR; in the CRISPR method, crRNA targets target nucleic acid, and the false positive problem brought by RPA is eliminated with high specificity; the magnitude of the CRISPR trans-effect cutting probe expands the signal, and solves the problem of low sensitivity (false negative) of the traditional colloidal gold test paper technology; the detection result presented by the colloidal gold test paper is simple and easy to understand, and is immediately visible.
The foregoing description is only illustrative of the present application and is not intended to limit the scope of the application, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present application.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute of China academy of sciences
<120> An immediate detection system and method for pathogen nucleic acid
<160> 21
<170> PatentIn version 3.3
<210> 1
<211> 20
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<213> Artificial sequence
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gattagcgta cgcacgttac 20
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<212> DNA
<213> Artificial sequence
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gctaatcg 8
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<212> RNA
<213> Artificial sequence
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aauuucuacu guuguagauc cagacauuuu gcucuca 37
<210> 4
<211> 37
<212> RNA
<213> Artificial sequence
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aauuucuacu guuguagauc aagacucacg uuaacaa 37
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<212> RNA
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aauuucuacu guuguagauc cagaaacuuu gcucuca 37
<210> 6
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<213> Artificial sequence
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aauuucuacu guuguagauc cagacucaag ggcuugu 37
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aauuucuacu guuguagauc aguugcuucg aauguua 37
<210> 8
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aauuucuacu guuguagaug gucgcccucu gauuagu 37
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aauuucuacu guuguagauc aacgucguga cugggaa 37
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caagaaattc aactccaggc agcagtaggg gaac 34
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<212> DNA
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ctttagtggc agtacgtttt tgccgaggct tct 33
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tactcattcg tttcggaaga gacaggtacg tt 32
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cccagtcacg acgttgtaaa acg 23
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agcggataac aatttcacac agg 23
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aagggcgatc ggtgcgggcc tcttcgctat tac 33
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cggctcgtat gttgtgtgga attgtgagcg gat 33
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gatcactaat acgactcact atagggcatc acgtagtcgc ggtaattcaa gaaattcaac 60
tcctggcagc agtaggggaa attctcctgc tcgaatggct agcggaggtg gtgaaactgc 120
cctcgcgcta ttgctgctag acagattgaa ccagcttgag agcaaagttt ctggtaaagg 180
ccaacaacaa caaggccaaa ctgtcactaa gaaatctgct gctgaggcat ctaaaaagcc 240
tcgccaaaaa cgtactgcca caaaacagta caacgtcact caagcatttg ggagacgtgg 300
tccagaacaa acccaaggaa atttcgggga ccaagaccta atcagacaag gaactgatta 360
caaacattgg ccgcaaattg cacaatttgc tccaagtgcc tctgcattct ttggaatgtc 420
acgcat 426
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<212> DNA
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gatcactaat acgactcact ataggggcag aaactcttcc agatctagtt cacaaggttc 60
aagatcagga aactctaccc gcggcacttc tccaggtcca tctggaatcg gagcagtagg 120
aggtgatcta ctttaccttg atcttctgaa cagactacaa gcccttgagt ctggcaaagt 180
aaagcaatcg cagccaaaag taatcactaa gaaagatgct gctgctgcta aaaataagat 240
gcgccacaag cgcacttcca ccaaaagttt caacatggtg caagcttttg gtcttcgcgg 300
accaggagac ctccagggaa actttggtga tcttcaattg aataaactcg gcactgagga 360
cccacgttgg ccccaaattg ctgagcttgc tcctacagcc agtgctttta tgggtatgtc 420
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gatcactaat acgactcact atagggtcaa gatacagcaa gaagttcaag ccggaaatag 60
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agccgggaga caaaataaca ttcgaagcaa ctggaaatct agtggtaccg agatatgcat 180
tcgcaatgga aagaaatgct ggatctggta ttatcatttc agatacacca gtccacgatt 240
gcaatacaac ttgtcaaaca cccaagggtg ctataaacac cagcctccca tttcagaata 300
tacatccgat cacaattgga aaatgtccaa aatatgtaaa aagcacaaaa ttgagactgg 360
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aacggttttg agatgatttg ggatccgaac ggatggactg ggacagacaa taacttctca 180
ataaagcaag atatcgtagg aataaatgag tggtcaggat atagcgggag ttttgttcag 240
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Claims (3)
1. An on-the-fly detection system for a pathogen SARS-CoV2 nucleic acid, the system comprising:
a lysis buffer for inactivating lysis of a sample to be detected to release RNA, the lysis buffer comprising: 5M-6M guanidine hydrochloride, 50mM-150mM Tris-HCl, 25mM-100mM EDTA, 100mM-200mM sodium chloride, 0.5mM DTT, and the balance water;
RT premix solution for reverse transcription of RNA in the sample into DNA, wherein the reverse transcription time is 10 minutes, and the reverse transcription reaction temperature is 37 ℃; the RT premix comprises: RT-RPA primer F, RT-RPA primer R, reaction buffer, RNase inhibitor, dNTP, reverse transcriptase and ultra-pure water; wherein, the concentration of RT-RPA primer F is 0.3uM-0.6uM; the concentration of RT-RPA primer R is 0.3uM-0.6uM;
the RPA premix is used for carrying out RPA reaction on the sample treated by the RT premix to amplify DNA in the sample, wherein the reaction time of the RPA reaction is 30 minutes, and the amplification temperature is 37 ℃; the RPA premix comprises: RT-RPA primer F, RT-RPA primer R, reaction buffer, mgOAc and ultrapure water; wherein, the concentration of RT-RPA primer F is 0.5uM-1.0uM; the concentration of RT-RPA primer R is 0.5uM-1.0uM;
The CRISPR reaction premix is used for carrying out specific trans-cleavage reaction on the sample treated by the RPA premix, and comprises Cas12a protein, crRNA, a probe, a reaction buffer solution and ultrapure water;
The colloidal gold test paper is used for developing the color of the sample treated by the CRISPR reaction premix solution so as to confirm whether the sample contains the pathogen nucleic acid or not, and the color development time of the colloidal gold test paper is 5 minutes;
The probe is an FB-ssDNA probe or an FQ-ssDNA probe; the sequence of the FB-ssDNA probe is 5'-FITC-GATTA GCGTA CGCAC GTTAC-Biotin-3'; the FQ-ssDNA probe consists of single-stranded DNA and a second group and a third group marked at the end part of the single-stranded DNA, wherein the second group is fluorescein isothiocyanate FITC or fluorescein isothiocyanate FITC and 6-carboxyfluorescein FAM, the third group comprises a black hole quenching group BHQ1, the third group is used for absorbing fluorescence, and when the CRISPR reaction premix solution does not detect target nucleic acid in a sample, the CRISPR reaction premix solution can not cut the probe, and the complete probe is marked with two groups at the same time; when the CRISPR reaction premix detects target nucleic acid in a sample, the trans-cleavage activity is exerted to cleave the probe into two or more sections so as to separate two groups, and the base sequence of the single-stranded DNA is shown as SEQ ID NO.1 or as SEQ ID NO. 2;
The amplification site of SARS-CoV2 is the sequence of crRNA corresponding to SARS-CoV 2N gene: 5'-AAUUU CUACU GUUGU AGAU ccaga cauuu ugcuc uca-3' the sequence of the corresponding RT-RPA primer F is: 5'-CAAGA AATTC AACTC CAGGC AGCAG TAGGG GAAC-3', sequence of RT-RPA primer R is: 5'-CTTTA GTGGC AGTAC GTTTT TGCCG AGGCT TCT-3';
And, the amplification site of SARS-CoV2 is SARS-CoV 2E gene, the corresponding crRNA sequence is: 5'-AAUUU CUACU GUUGU AGAU caaga cucac guuaa caa-3', the sequence of the corresponding RT-RPA primer F is as follows: 5'-TACTC ATTCG TTTCG GAAGA GACAG GTACG TT-3'; the sequence of the RT-RPA primer R is as follows: 5'-CAGAT TTTTA ACACG AGAGT AAACG TAAAAAGAA-3'.
2. The system of claim 1, wherein crRNA is present at a concentration of 0.5uM to 1uM and cas12a protein is present at a concentration of 0.1uM to 0.5uM in the mixture of CRISPR reaction premix and the sample partially after the RPA premix; when the probe is a FB-ssDNA probe, the concentration is 0.5nM to 2.0nM; when the probe is FQ-ssDNA probe, its concentration is 1nM-10nM.
3. A method for the on-the-fly detection of a pathogen SARS-CoV2 nucleic acid for non-disease diagnosis and treatment, the method utilizing the system of any one of claims 1-2 to detect a pathogen SARS-CoV2 nucleic acid, the method comprising: placing a sample to be detected in the lysis buffer solution, inactivating and lysing the sample to release RNA; placing the sample comprising RNA in the RT premix to reverse transcribe the RNA into DNA; placing the sample subjected to reverse transcription in the RPA premix solution to amplify the sample; placing the amplified partial sample in the CRISPR reaction premix to perform a specific cleavage reaction; and developing the color of the sample subjected to the specific cleavage reaction by using the colloidal gold test paper.
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| CN114045354B (en) * | 2021-11-15 | 2024-07-12 | 上海博满生物科技有限公司 | One-step real-time detection method for direct-amplification ERA coupled CRISPR/cas12a nucleic acid |
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