A method and application for rapid and accurate detection of African swine fever virus based on CRISPR/Cas12a

An African swine fever virus and precise technology, applied in the field of pathogen detection, can solve the problems of inability to determine positive and negative sample detection and accurate judgment, unknown negative and positive samples, prevention and control, etc., to achieve early detection of ASF, increased sensitivity, and accurate detection Effect

Active Publication Date: 2021-09-07
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A method and application for rapid and accurate detection of African swine fever virus based on CRISPR/Cas12a
  • A method and application for rapid and accurate detection of African swine fever virus based on CRISPR/Cas12a
  • A method and application for rapid and accurate detection of African swine fever virus based on CRISPR/Cas12a

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Experimental program
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Effect test

Embodiment 1

[0043] The design of embodiment 1 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 for ASFV VP72 gene (GenBank accession no. MH766894.1), and prepared 19 crRNAs (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 into a 200 μl centrifuge tube, mix well and centrifuge. Put it into a PCR machine for annealing reaction: 37°C, 30min; 95°C, 5min; drop 5°C per minute, to 25°C, to obtain Template DNA. ssDNA sense and ssDNA antisense were synthesized by Shanghai Sangong.

[0050] (2) Transcription

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

[0052] Table 2

[...

Embodiment 2

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

[0058] 1. Target fragment amplification

[0059] (1) Extraction of viral DNA

[0060] 200 μL of serum samples were taken and treated with RaPure Viral RNA / DNA Kit (Magen, China) according to the operation steps to extract viral nucleic acids.

[0061] (2) RPA amplification

[0062] RPA-F / RPA-R designed for RPA amplification, the sequence 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 the 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, put the reaction tube into a 37°C constant temperature incubator and incubate for 10min to complete the RPA amplification. RPA amplification pro...

Embodiment 3

[0072] Example 3 The crRNA with the highest screening efficiency

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

[0074] The result is as figure 1 , figure 2 As shown: after the reaction time of 15 and 30 minutes, 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 and application for rapid and accurate detection of African swine fever virus based on CRISPR / Cas12a. Based on the research of the present invention, when the ASF virus nucleic acid forms a ternary complex with Cas12a and crRNA, the RuvC domain of Cas12a in the complex performs DNase activity and cuts single-stranded DNA labeled with a fluorescent signal. Check whether the sample contains ASF virus nucleic acid. And designed high-quality crRNA, built a method for rapid and accurate detection of African swine fever virus. It has excellent detection effect on ASF virus, strong specificity, high accuracy, and the sensitivity is more than 10 times higher than that of fluorescent quantitative PCR. The reaction time is shorter and it does not need to be expensive. In addition, for weakly positive and suspected positive samples with extremely low virus content, it is also possible to determine whether the sample contains ASFV by appropriately extending the reaction time. The method of the invention can efficiently and accurately detect African swine fever virus, has the advantages of simple operation and no need for professional laboratories, and provides an effective method for the first-line rapid detection of the virus in pig farms, and is of great significance for early monitoring, diagnosis, prevention and control of 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 pigs and wild boars. The acute type manifests as high fever, depression, Anorexia, skin cyanosis, splenomegaly and hemorrhage, the morbidity and mortality are as high as 100%. Since African swine fever entered China in August 2018, the disease has rapidly swept across the country, causing huge economic losses. The country's reproductive sows have declined sharply, prompting subversive changes in my country's pig industry and animal health-related enterprises. There is currently no vaccine for this disease, and there is no effective anti-ASF virus drug, so early monitor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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