Development of a reverse transcription loop-mediated isothermal amplification based clustered regularly interspaced short palindromic repeats Cas12a assay for duck Tembusu virus

Yangbao Ding; Zhanhong Huang; Xinbo Li; Mei Tang; Weiqiang Li; Siyu Feng; Luxiang Zhao; Junsheng Zhang; Shichao Yuan; Fen Shan; Peirong Jiao

doi:10.3389/fmicb.2023.1301653

Development of a reverse transcription loop-mediated isothermal amplification based clustered regularly interspaced short palindromic repeats Cas12a assay for duck Tembusu virus

Front Microbiol. 2023 Nov 30:14:1301653. doi: 10.3389/fmicb.2023.1301653. eCollection 2023.

Authors

Yangbao Ding^{1

2

3}, Zhanhong Huang¹, Xinbo Li¹, Mei Tang¹, Weiqiang Li¹, Siyu Feng¹, Luxiang Zhao¹, Junsheng Zhang¹, Shichao Yuan¹, Fen Shan⁴, Peirong Jiao^{1

2

3}

Affiliations

¹ College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China.
² Key Laboratory of Animal Vaccine Development, Ministry of Agriculture and Rural Affairs, Guangzhou, China.
³ Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, Guangzhou, China.
⁴ Guangzhou Collaborative Innovation Center on Science-Tech of Ecology and Landscape, Guangzhou Zoo, Guangzhou, China.

Abstract

Duck Tembusu virus (DTMUV) is an emerging pathogen that poses a serious threat to the duck industry in China. Currently, polymerase chain reaction (PCR), quantitative PCR (qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) are commonly used for DTMUV detection. However, these methods require complex steps and special equipment and easily cause false-positive results. Therefore, we urgently need to establish a simple, sensitive and specific method for the clinical field detection of DTMUV. In this study, we developed an RT-LAMP-based CRISPR-Cas12a assay targeting the C gene to detect DTMUV with a limited detection of 3 copies/μL. This assay was specific for DTMUV without cross-reaction with other common avian viruses and only required some simple pieces of equipment, such as a thermostat water bath and blue/UV light transilluminator. Furthermore, this assay showed 100% positive predictive agreement (PPA) and negative predictive agreement (NPA) relative to SYBR Green qPCR for DTMUV detection in 32 cloacal swabs and 22 tissue samples, supporting its application for clinical field detection.

Keywords: CRISPR-Cas12a; RT-LAMP; Tembusu virus; clinical field detection; duck.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was funded by grant from the Laboratory of Lingnan Modern Agriculture Project (NT2021007).