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Method for rapidly and precisely detecting African swine fever virus on basis of CRISPR/Cas12a and application

A kind of African swine fever virus and precise technology, applied in the field of pathogen detection, can solve the problems of difficult and indeterminate negative and positive sample detection and accurate judgment, prevention and control, etc., achieve short detection time, facilitate early and rapid detection, and low cost Effect

Active Publication Date: 2019-11-15
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, early monitoring and prevention are extremely important for the prevention and control of African swine fever. Once the African swine fever virus has formed a certain scale, even if it is detected, it is difficult to have a good means of complete prevention and control
Therefore, for the prevention and control of African swine fever virus, it is very critical for the successful detection of samples with very low virus content that cannot be determined to be negative or positive for viral nucleic acid. This is affected by many factors. At present, the method developed by the team of Professor Doudna above has not yet Carry out detailed research, that is, whether this technology can be applied to the detection of weak positive and difficult / unable to determine negative and positive samples, and accurately determine the positive and negative of samples is unknown, and there is no relevant in-depth research

Method used

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  • Method for rapidly and precisely detecting African swine fever virus on basis of CRISPR/Cas12a and application
  • Method for rapidly and precisely detecting African swine fever virus on basis of CRISPR/Cas12a and application
  • Method for rapidly and precisely detecting African swine fever virus on basis of CRISPR/Cas12a and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Design of crRNA

[0044] The core of the CRISPR / Cas12a detection method lies in crRNA, so the quality of crRNA is directly related to the sensitivity and accuracy of the detection method. In order to screen the crRNA with the highest efficiency, we designed 19 ssDNAs against ASFV VP72 gene (GenBank accessionno.MH766894.1), and prepared 19 crRNAs (the sequences are shown in SEQ ID NO.1-19).

[0045] (1) Annealing system as shown in Table 1

[0046] Table 1

[0047]

[0048]

[0049] Add the above components to a 200 μl centrifuge tube, mix well and centrifuge. Put into a PCR machine for annealing reaction: 37°C, 30min; 95°C, 5min; drop 5°C every minute to 25°C to obtain Template DNA. ssDNA sense and ssDNA antisense were synthesized by Shanghai Shenggong.

[0050] (2) Transcription

[0051] with MEGAshortscript TM T7 Transcription Kit (Invitrogen, USA) was used for transcription, and the reaction system was shown in Table 2:

[0052] Table 2

[00...

Embodiment 2

[0057] Example 2 Establishment of CRISPR / Cas12a detection method for ASFV

[0058] 1. Amplification of the target fragment

[0059] (1) Extract viral DNA

[0060] Take 200 μL of serum samples and use RaPure Viral RNA / DNA Kit (Magen, China) to process according to the operation steps to extract viral nucleic acid.

[0061] (2) RPA amplification

[0062] The RPA-F / RPA-R designed for RPA amplification is shown in SEQ ID NO. 20-21.

[0063] The RPA amplification system is shown in Table 3

[0064] table 3

[0065]

[0066] Add 46 μL of the above mixture to a Twist Amp NFO kit (TwistDxTM, Cambridge, United Kingdom) reaction tube containing lyophilized enzyme powder, pipette the lyophilized enzyme powder until it is completely dissolved, and then add 4 μL of magnesium acetate solution to the reaction tube. , Mix well, and put the reaction tube into a 37°C constant temperature incubator for 10 min to complete the RPA amplification. The RPA amplification products were subject...

Embodiment 3

[0072] Example 3 Screening crRNA with the highest efficiency

[0073] RaPure Viral RNA / DNA Kit (Magen, China) was used for 2 clinical samples of ASF virus nucleic acid positive samples, and the virus nucleic acid was extracted according to the operation steps. Then, according to the RPA amplification method and conditions in Example 1, RPA amplification was carried out. Finally, the reaction was carried out according to the CRISPR / Cas12a detection method and conditions of ASFV in Example 1. The 19 crRNAs prepared above were used as the crRNAs in the reaction system, and the reaction time was 15 or 30 min. After the reaction, the fluorescence value was read with a BioTek microplate reader.

[0074] The result is as figure 1 , figure 2 Shown: after the reaction time of 15 and 30 min, the fluorescence values ​​of crRNA5 and crRNA15 are the highest, indicating that among the 19 crRNAs, these two have the best effect and the highest sensitivity.

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Abstract

The invention discloses a method for rapidly and precisely detecting an African swine fever virus on the basis of CRISPR / Cas12a and application. On the basis of the research of the method, when ASF virus nucleic acid forms a ternary complex with Cas12a and crRNA, a RuvC structural domain of Cas12a in the complex exerts DNase activity, single-stranded DNA with a fluorescent signal label is cut, whether a sample to be detected contains the ASF virus nucleic acid or not can be known by detecting fluorescence, and high-quality crRNA is designed, so that the method for rapidly and precisely detecting the African swine fever virus is constructed. The method has the advantages that the ASF virus detection effect is excellent, the specificity is strong, the precision is high, the sensitivity is 10times higher than that of a fluorescent quantitative PCR, the reaction time is shorter, and expensive instruments are not needed. In addition, for weak positive and suspected positive samples with extremely low virus content, whether or not the samples contain the ASFV can also be determined by properly prolonging the reaction time. The method can be used for efficiently and precisely detecting the African swine fever virus and has the advantages of simple operation, no need of a professional laboratory and the like; the effective method is provided for first-line rapid detection of the virusin a pig farm and has important significance in early monitoring, diagnosis and prevention and control of the African swine fever.

Description

technical field [0001] The invention belongs to the technical field of pathogen detection. More specifically, it relates to a method and application for rapid and accurate detection of African swine fever virus based on CRISPR / Cas12a. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contagious infectious disease caused by African swine fever virus (ASFV) infecting domestic and wild boars. Anorexia, skin cyanosis, spleen hemorrhage, morbidity and mortality up to 100%. Since African swine fever entered China in August 2018, the disease has rapidly swept across the country, causing huge economic losses and a sharp decline in reproductive sows across the country, prompting subversive changes in my country's pig industry and animal health-related enterprises. At present, there is no vaccine for this disease, and there is no effective drug to inhibit African swine fever virus. Therefore, early monitoring and diagnosis and biosafety are very im...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2521/301C12Q2565/625C12Q2563/107
Inventor 郭春和赵娜贺胜王小瑛王刚刘小红陈瑶生
Owner SUN YAT SEN UNIV
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