Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever.

Jing Bai,Hongli Du,Junshan Liu,Guorui Zhong,Yang Zhou,Jinyi Yang,Weifan Jiang,Haojian Li,Luting Wu,Qingmei Xie,Lizhen Huang,Haosi Lin

doi:10.3389/fmicb.2019.02830

Abstract

The mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and wild boars is caused by African swine fever virus (ASFV), can reach 100%. Since the first confirmed ASF outbreak in China on 3 August 2018, 156 ASF outbreaks were detected in 32 provinces. About 1,170,000 pigs were culled in order to halt further spread. 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 and the assay displayed no cross-reactivity to other 13 swine viruses including classical swine fever (CSF). CORDS could identify the ASFV DNA target at femtomolar sensitivity in an hour at 37°C, and only requires an incubator. For ease of use, the reagents of CORDS were lyophilized to three tubes and remained the same sensitivity when stored at 4°C for at least 7 days. Thus, CORDS provide 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-based applications.

Highlights

African swine fever (ASF) is a highly contagious hemorrhagic disease of domestic pigs and wild boars
ASF occurred as a rapid outbreak across the nation, indicating its highly contagious nature (Food and Agricultural Organization of the United Nations, 2019)
Given its similar clinical signs to several other porcine diseases (Galindo and Alonso, 2017) and the lack of laboratory facilities for diagnosis on farms, diagnosing ASF often requires infected samples to be transported to a qualified laboratory, which delays diagnosis and increases the chances of contagion

Summary

Introduction

African swine fever (ASF) is a highly contagious hemorrhagic disease of domestic pigs and wild boars. The molecular tools for detecting ASFV, such as polymerase chain reaction (PCR) and reverse-transcriptase (RT)-PCR, have become popular because of their sensitivity and specificity for ASF diagnosis (Oura et al, 2013) Such methods are not suitable for on-site situations (e.g., on farms) or for rapid viral detection as they require thermal cycling instruments and skilled operators (Ikeno et al, 2013; Boonham et al, 2014; Khodakov et al, 2016). Like other amplification technologies, its resolution depends on the binding specificity between the primers and templates, which can limit their accuracy (Boonham et al, 2014; He et al, 2019)

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Results

Conclusion