Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics

Shuhan Lu,Qiubing Chen,Junyi Duan,Xinlin Lei,Dongming Zhao,Hao Yin,Jing Wu,Zhigao Bu,Fang Li,Ying Zhang

doi:10.1038/s41421-020-0151-5

Abstract

African swine fever virus (ASFV) is a dsDNA virus responsible for a severe, highly contagious, and lethal disease affecting both domestic and wild pigs. ASFV has brought enormous economic loss to a number of countries, and effective vaccine and therapy are still lacking. Therefore, a rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we developed a Cas12a-mediated portable paper assay to rapidly and precisely detect ASFV. We identified a robust set of crRNAs that recognized the highly conserved region of essential ASFV genes. The Cas12a-mediated detection assay showed low tolerance for mismatch mutations, and no cross-reactivity against other common swine pathogens. We further developed a paper-based assay to allow instrument-free detection of ASFV. Specifically, we applied gold nanoparticle–antibody conjugate to engineer homemade strips and combined it with Cas12a-mediated ASFV detection. This portable paper, instrument-free diagnostics, faithfully detected ASFV in swine samples, showing comparable sensitivity to the traditionally instrument-dependent qPCR method. Taking together, we developed a highly sensitive, instant, and economic Cas12a-mediated paper diagnostics of ASFV, with a great application potential for monitoring ASFV in the field.

Highlights

African swine fever (ASF) is a highly contagious disease of swine that poses enormous economic losses due to its high mortality rate and rapid spread[1,2]
Select robust crRNA against the conservative regions of ASF virus (ASFV) The crRNA design has played an essential role in the sensitivity of Cas12a-based viral detection[21,22,23,24]
We designed six crRNAs by either targeting the conserved regions of polyprotein pp[220] or DNA polymerase gene (DNA Pol), both of which are essential to ASFV life cycle[2,5] (Supplementary Table S1)

Summary

Introduction

African swine fever (ASF) is a highly contagious disease of swine that poses enormous economic losses due to its high mortality rate and rapid spread[1,2]. ASF has spread into a number of countries in Africa, Europe, and Asia during the past decade, with the possibility of further expansion[1,3]. Due to the lack of vaccine and effective treatments against ASFV, disease control mainly relies on culling pigs[1,4]. More than 30 million domestic pigs were culled in the past 2 years, and the number continues to increase[6]. Rapid diagnosis of the ASFV-affected animal is crucial to prevent its broad expansion, and to reduce economic losses[7,8]

Objectives

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Results

Conclusion