CN115927764A - Respiratory virus detection method based on CRISPR - Google Patents
Respiratory virus detection method based on CRISPR Download PDFInfo
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- CN115927764A CN115927764A CN202310016823.2A CN202310016823A CN115927764A CN 115927764 A CN115927764 A CN 115927764A CN 202310016823 A CN202310016823 A CN 202310016823A CN 115927764 A CN115927764 A CN 115927764A
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- 238000001514 detection method Methods 0.000 title claims abstract description 56
- 241000700605 Viruses Species 0.000 title claims abstract description 34
- 230000000241 respiratory effect Effects 0.000 title claims abstract description 15
- 108091033409 CRISPR Proteins 0.000 title claims abstract description 10
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract description 10
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 26
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 26
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 26
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 241000725643 Respiratory syncytial virus Species 0.000 claims abstract description 8
- 241000712461 unidentified influenza virus Species 0.000 claims abstract description 8
- 238000012795 verification Methods 0.000 claims abstract description 7
- 238000012545 processing Methods 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 10
- 238000002474 experimental method Methods 0.000 claims description 6
- 239000000523 sample Substances 0.000 abstract description 32
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 abstract description 6
- 238000010362 genome editing Methods 0.000 abstract description 4
- 238000012408 PCR amplification Methods 0.000 abstract description 3
- 238000009396 hybridization Methods 0.000 abstract description 3
- 108700004991 Cas12a Proteins 0.000 description 6
- 108091027544 Subgenomic mRNA Proteins 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 4
- 230000003252 repetitive effect Effects 0.000 description 4
- 108020000999 Viral RNA Proteins 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 241000711573 Coronaviridae Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 238000003199 nucleic acid amplification method Methods 0.000 description 1
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- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000012123 point-of-care testing Methods 0.000 description 1
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- 241000701161 unidentified adenovirus Species 0.000 description 1
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- 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
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a respiratory virus detection method based on CRISPR, and relates to the technical field of virus detection. S1: establishing and optimizing a sample processing system; s2: establishing a nucleic acid detection system based on cas12 a; s3: establishing detection methods of influenza virus, respiratory syncytial virus and the like based on cas12 a; s4: verifying a positive sample; s5: extracting nucleic acid; s6: detecting viruses; s7: verifying a clinical sample; performing clinical sample verification on the positive sample subjected to PCR detection and cas12 virus detection in the step S6; s8: detecting a field under a severe cold condition; and (3) detecting the sample after clinical sample verification under a severe cold condition. The SHERLOR utilizes the RNA hybridization and gene editing technology to establish the quasi-nucleic acid detection technology, the technology uses unpurified sample nucleic acid to carry out PCR amplification, reduces the nucleic acid loss in the nucleic acid extraction process, improves the accuracy and simultaneously ensures the sensitivity, and the sensitivity of SHERLOCK detection can be improved by using a plurality of crRNA probes.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to a respiratory virus detection method based on CRISPR.
Background
Respiratory viruses are important viruses causing major public health events, common pathogenic viruses comprise influenza viruses, respiratory syncytial viruses, adenoviruses, novel coronaviruses and the like, the viruses have longer life cycle in a cold environment, and researches show that the viruses can survive for more than 20 years in an environment at the temperature of-20 ℃. China has a large number of cold regions in the north and the west, and has potential conflict possibility. Even if a field rapid detection technology is established, the treatment capacity of dealing with biological safety events in cold regions including respiratory viruses can be improved. Respiratory viruses have high morbidity, are rapidly transmitted and are very easy to cause serious public safety incidents, and a rapid and accurate detection method is needed to achieve the aims of early discovery, early isolation and early treatment.
Disclosure of Invention
The invention aims to provide a CRISPR-based respiratory virus detection method, which improves the detection sensitivity of a SHERLLOCK technology by using a plurality of crRNA probes through a CRISPR system SHERLLOCK technology, thereby directly measuring the RNA of a virus without carrying out reverse transcription PCR. The detection signal output based on the CRISPR system can be used for visual detection and fluorescent signal detection, so that the rapid detection of the POCT or battlefield virus is realized. Aiming at the technical problem of on-site instant detection of important respiratory viruses in cold regions, the invention mainly solves the short-plate problem of on-site instant detection of important respiratory viruses in cold regions and realizes rapid detection of respiratory pathogens.
The technical purpose of the invention is realized by the following technical scheme: a CRISPR-based respiratory virus detection method comprises the following process steps:
s1: establishing and optimizing a sample processing system;
s2: establishing a nucleic acid detection system based on cas12 a;
s3: establishing detection methods of influenza virus, respiratory syncytial virus and the like based on cas12 a;
s4: verifying a positive sample;
s5: extracting nucleic acid; dividing the positive samples verified in the step S4 into two groups, and respectively adopting a control method and an experimental method to carry out nucleic acid extraction;
s6: detecting viruses; performing PCR detection on the positive sample subjected to nucleic acid extraction by adopting a control method in the step S5; performing a cas 12-based virus detection method on the positive sample subjected to nucleic acid extraction by using the experimental method in the step S5;
s7: verifying a clinical sample; performing clinical sample verification on the positive sample subjected to PCR detection and cas12 virus detection in the step S6;
s8: detecting a severe cold condition field; and (3) detecting the sample after clinical sample verification under a severe cold condition.
In conclusion, the invention has the following beneficial effects: the SHERLOR utilizes the RNA hybridization and gene editing technology to establish the quasi-nucleic acid detection technology, the technology can use unpurified sample nucleic acid to carry out PCR amplification, the nucleic acid loss in the nucleic acid extraction process is reduced, the accuracy is improved, meanwhile, the sensitivity is ensured, the sensitivity of SHERLLOCK detection can be improved by using a plurality of crRNA probes, and therefore, the PCR is not needed, and the virus DNA or RNA is directly measured. Can be applied to the emergency response and defense capability of public safety events caused by cold regions, particularly respiratory viruses.
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FIG. 1 is a flow chart of the steps of an embodiment of the present invention.
Detailed Description
The present invention is described in further detail below with reference to FIG. 1.
Example (b): a CRISPR-based respiratory virus detection method, as shown in fig. 1, comprising the following process steps:
s1: establishing and optimizing a sample processing system; SHERLLOCK is an accurate nucleic acid detection technology established by RNA hybridization and gene editing technology; the technology adopts unpurified sample nucleic acid to carry out PCR amplification, reduces the nucleic acid damage in the nucleic acid extraction process, improves the accuracy and ensures the sensitivity; by using multiple crRNA probes, the sensitivity of SHERLock detection can be improved, thereby directly determining viral DNA or RNA without PCR.
S2: establishing a nucleic acid detection system based on cas12 a; constructing an expression vector of the Cas12a protein, optimizing protein expression conditions, expressing and purifying the Cas12a, and obtaining the high-purity Cas12a protein; constructing an sgRNA in-vitro transcription vector, establishing and optimizing a transcription system of the sgRNA, reconstructing a compound of Cas12a and the sgRNA, and detecting the nuclease activity of the sgRNA.
S3: establishing detection methods of influenza virus, respiratory syncytial virus and the like based on cas12 a; screening specific candidate detection sites of influenza virus and respiratory syncytial virus; the detection method for influenza virus and respiratory syncytial virus is constructed, visual signal detection and fluorescent signal detection are respectively tested by optimizing the concentration of Cas12a and sgRNA and the composition of reaction buffer solution, the sensitivity of two signal supply modes is compared, and the detection method for influenza virus and respiratory syncytial virus based on Cas12a is determined.
S4: verifying a positive sample;
s5: extracting nucleic acid; dividing the positive samples verified in the step S4 into two groups, and respectively adopting a control method and an experimental method to extract nucleic acid; extracting viral RNA from the specimen, and reverse-transcribing the viral RNA into single-stranded cDNA using an enzyme reverse transcriptase; amplification of the PCR product is coupled with fluorescence detection of the labeled PCR product.
S6: virus detection; performing PCR detection on the positive sample subjected to nucleic acid extraction by adopting a control method in the step S5; performing a cas 12-based virus detection method on the positive sample subjected to nucleic acid extraction by using the experimental method in the step S5; artificially constructing an interval repetitive sequence for specifically recognizing cell genome, combining the interval repetitive sequence with cas9 protein to form a compound, cutting a target genome, performing homologous recombination and repair by using the DNA repair capability of the cell genome to obtain a corresponding cell with accurate gene editing, adding the identification sequence of the interval repetitive sequence into the system after the interval repetitive sequence and cas 12-day protein form the compound, and obtaining the activity of non-specific RNA enzyme by a nucleoprotein compound so as to degrade RNA in the system without difference. The method is a brand-new isothermal amplification technology, has strong specificity, can be used for realizing different signal outputs by determining and combining various signal supply modes, and can be used for fluorescent signals and visual detection.
S7: verifying a clinical sample; performing clinical sample verification on the positive sample subjected to PCR detection and cas12 virus detection in the step S6;
s8: detecting a severe cold condition field; and (3) detecting the clinical sample verified sample under a severe cold condition.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (1)
1. A respiratory virus detection method based on CRISPR is characterized in that: the method comprises the following steps:
s1: establishing and optimizing a sample processing system;
s2: establishing a nucleic acid detection system based on cas12 a;
s3: establishing detection methods of influenza virus, respiratory syncytial virus and the like based on cas12 a;
s4: verifying a positive sample;
s5: extracting nucleic acid; dividing the positive samples verified in the step S4 into two groups, and respectively adopting a control method and an experimental method to extract nucleic acid;
s6: virus detection; performing PCR detection on the positive sample subjected to nucleic acid extraction by adopting a control method in the step S5; performing a cas 12-based virus detection method on the positive sample subjected to the nucleic acid extraction by the experimental method in the step S5;
s7: verifying a clinical sample; performing clinical sample verification on the positive sample subjected to PCR detection and cas12 virus detection in the step S6;
s8: detecting a severe cold condition field; and (3) detecting the sample after clinical sample verification under a severe cold condition.
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| CN202310016823.2A CN115927764A (en) | 2023-01-06 | 2023-01-06 | Respiratory virus detection method based on CRISPR |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111778357A (en) * | 2020-07-13 | 2020-10-16 | 国家卫生健康委科学技术研究所 | CRISPR/Cas12 a-based respiratory syncytial virus nucleic acid rapid detection kit and detection method thereof |
| CN113373264A (en) * | 2021-06-10 | 2021-09-10 | 安徽医科大学 | Novel coronavirus double-target rapid detection method and kit based on CRISPR/Cas system |
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- 2023-01-06 CN CN202310016823.2A patent/CN115927764A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111778357A (en) * | 2020-07-13 | 2020-10-16 | 国家卫生健康委科学技术研究所 | CRISPR/Cas12 a-based respiratory syncytial virus nucleic acid rapid detection kit and detection method thereof |
| CN113373264A (en) * | 2021-06-10 | 2021-09-10 | 安徽医科大学 | Novel coronavirus double-target rapid detection method and kit based on CRISPR/Cas system |
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