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

Autor: Yangbao Ding, Zhanhong Huang, Xinbo Li, Mei Tang, Weiqiang Li, Siyu Feng, Luxiang Zhao, Junsheng Zhang, Shichao Yuan, Fen Shan, Peirong Jiao
Jazyk: angličtina
Rok vydání: 2023
Předmět:
Zdroj: Frontiers in Microbiology, Vol 14 (2023)
Druh dokumentu: article
ISSN: 1664-302X
DOI: 10.3389/fmicb.2023.1301653
Popis: 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.
Databáze: Directory of Open Access Journals