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bioRxiv · 10.1101/729590

Cas12a-based on-site and rapid nucleic acid detection of African swine fever

Bai, J.·Lin, H.·Li, H.·Zhou, Y.·Liu, J.·Zhong, G.·Wu, L.·Jiang, W.·Du, H.·Yang, J.·Xie, Q.·Huang, L.
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Abstract

The mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and is caused by African swine fever virus (ASFV), can reach 100%. ASF has been reported in 25 Chinese provinces since August 2018. There is no effective treatment or vaccine for it and the present molecular diagnosis technologies have trade-offs in sensitivity, specificity, cost and speed, and none of them cater perfectly to ASF control. Thus, a technology that overcomes the need for laboratory facilities, is relatively low cost, and rapidly and sensitively detects ASFV would be highly valuable. Here, we describe an RAA-Cas12a-based system that combines recombinase-aided amplification (RAA) and CRISPR/Cas12a for ASFV detection. The fluorescence intensity readout of this system detected ASFV p72 gene levels as low as 10 aM. For on-site ASFV detection, lateral-flow strip readout was introduced for the first time in the RAA-Cas12a based system (named CORDS, Cas12a-based On-site and Rapid Detection System). We used CORDS to detect target DNA highly specifically using the lateral-flow strip readout. CORDS could identify the p72 gene at femtomolar sensitivity in an hour at 37{degrees}C, and only requires an incubator. For ease of use, the regents of CORDS was lyophilized to three tubes and remained the same sensitivity when stored at 4 {degrees}C for at least 7 days. Thus, CORDS provides a rapid, sensitive and easily operable method for ASFV on-site detection. Lyophilized CORDS can withstand long-term transportation and storage, and is ready for field applications.

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Bai, J., Lin, H., Li, H., Zhou, Y., Liu, J., Zhong, G., Wu, L., Jiang, W., Du, H., Yang, J., Xie, Q., Huang, L.. 2019-08-08. Cas12a-based on-site and rapid nucleic acid detection of African swine fever. https://doi.org/10.1101/729590

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