A one-tube rapid visual CRISPR assay for the field detection of Japanese encephalitis virus

Bingrong Xu; Ping Gong; Yi Zhang; Yuan Wang; Dagang Tao; Lanting Fu; Emmanuel M Khazalwa; Hailong Liu; Shuhong Zhao; Xuying Zhang; Shengsong Xie

doi:10.1016/j.virusres.2022.198869

A one-tube rapid visual CRISPR assay for the field detection of Japanese encephalitis virus

Virus Res. 2022 Oct 2:319:198869. doi: 10.1016/j.virusres.2022.198869. Epub 2022 Jul 13.

Authors

Bingrong Xu¹, Ping Gong², Yi Zhang², Yuan Wang¹, Dagang Tao¹, Lanting Fu¹, Emmanuel M Khazalwa³, Hailong Liu¹, Shuhong Zhao⁴, Xuying Zhang⁵, Shengsong Xie⁶

Affiliations

¹ Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, PR China.
² Institute of Animal Husbandry and Veterinary, Wuhan Academy of Agricultural Science, Wuhan 430208, PR China.
³ International Livestock Research Institute (ILRI), P.O. Box 30709, Nairobi 00100, Kenya.
⁴ Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, PR China; Hubei Hongshan Laboratory, Frontiers Science Center for Animal Breeding and Sustainable Production, Wuhan 430070, PR China.
⁵ Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, Hannover 30559, Germany. Electronic address: [email protected].
⁶ Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, PR China; Hubei Hongshan Laboratory, Frontiers Science Center for Animal Breeding and Sustainable Production, Wuhan 430070, PR China. Electronic address: [email protected].

PMID: 35842016
DOI: 10.1016/j.virusres.2022.198869

Abstract

Early and rapid detection of Japanese encephalitis virus (JEV) is necessary for timely preventive and control measures. However, JEV RNA detection remains challenging due to the low level of viremia. In this study, a RApid VIsual CRISPR (RAVI-CRISPR) assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and CRISPR/Cas12a targeting was developed for easy detection of JEV in the field. We showed successful detection of 8.97 or more copies of the C gene sequence of JEV RNA within approximately 60 min. This assay also displayed no cross-reactivity with other porcine pathogens. We applied our one-tube RAVI-CRISPR assay to 18 brain tissue sample for JE diagnosis. The results from both fluorescence intensity measurements and directly naked-eye visualization were consistent with those from real-time PCR analysis. Taken together, our results showed that one-tube RAVI-CRISPR assay is robust, convenient, sensitive, specific, affordable, and potentially adaptable to on-site detection or surveillance of JEV in clinical and vector samples.

Keywords: CRISPR/Cas12a; Japanese encephalitis virus; Naked-eye visualization; On-site detection; RT-LAMP.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Encephalitis Virus, Japanese* / genetics
Encephalitis, Japanese* / diagnosis
Nucleic Acid Amplification Techniques / methods
RNA
RNA, Viral / analysis
RNA, Viral / genetics
Reverse Transcription
Sensitivity and Specificity
Swine
Swine Diseases*

Substances

RNA, Viral
RNA