Rapid detection of Nipah virus using the one-pot RPA-CRISPR/Cas13a assay

Jing Miao; Lulu Zuo; Dongmei He; Zhixin Fang; Nicolas Berthet; Chao Yu; Gary Wong

doi:10.1016/j.virusres.2023.199130

Rapid detection of Nipah virus using the one-pot RPA-CRISPR/Cas13a assay

Virus Res. 2023 Jul 15:332:199130. doi: 10.1016/j.virusres.2023.199130. Epub 2023 May 14.

Authors

Jing Miao¹, Lulu Zuo¹, Dongmei He², Zhixin Fang³, Nicolas Berthet⁴, Chao Yu⁵, Gary Wong⁶

Affiliations

¹ Viral Hemorrhagic Fevers Research Unit, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shangha 200031, China; University of Chinese Academy of Sciences, Beijing 100049, China.
² Institute of Pathogenic Microorganisms, Guangdong Provincial Center for Disease Control and Prevention, Guangzhou 510440, China.
³ Biosafety Laboratory, Guangdong Second Provincial General Hospital, Guangzhou 510317, China.
⁴ Centre for Microbes, Development, and Health, Institut Pasteur of Shanghai, Unit of Discovery and Molecular Characterization of Pathogens, Chinese Academy of Sciences, Shanghai 200031, China; Institut Pasteur, Unité Environnement et Risque Infectieux, Cellule d'Intervention Biologique d'Urgence, Université Paris-cite, Paris 75724, France. Electronic address: nicolas.berthet@pasteur.fr.
⁵ Viral Hemorrhagic Fevers Research Unit, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shangha 200031, China. Electronic address: ychao@ips.ac.cn.
⁶ Viral Hemorrhagic Fevers Research Unit, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shangha 200031, China. Electronic address: garyckwong@ips.ac.cn.

Abstract

Nipah virus (NiV) is a zoonotic pathogen with airborne transmission and high case fatality rates in humans. There is currently no treatment or vaccine against NiV infection approved for humans or animals, therefore early diagnosis is the key to control any potential outbreaks. In this study, we developed an optimized one-pot assay using recombinase polymerase amplification (RPA) coupled to CRISPR/Cas13a for the molecular detection of NiV. The one-pot RPA-CRISPR/Cas13a assay for NiV detection was specific and did not cross-react against other selected (re)-emerging pathogens. The sensitivity of the one-pot RPA-CRISPR/Cas13a assay for NiV detection can detect as little as 10³ cp/μL of total synthetic NiV cDNA. The assay was then validated with simulated clinical samples. The results for the one-pot RPA-CRISPR/Cas13a assay could be visualized with either fluorescence or lateral flow strips for convenient clinical or field diagnostics, providing a useful supplement to the gold-standard qRT-PCR assay for detecting NiV detections.

Keywords: CRISPR/Cas13a; Lateral flow strips; Molecular detection; Nipah virus; Recombinase polymerase amplification.

Publication types

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

MeSH terms

Animals
Clustered Regularly Interspaced Short Palindromic Repeats
Humans
Nipah Virus* / genetics
Nucleic Acid Amplification Techniques / methods
Nucleotidyltransferases / genetics
Polymerase Chain Reaction / methods
Recombinases* / metabolism
Sensitivity and Specificity

Substances

Recombinases
Nucleotidyltransferases