Establishment of a RAA-CRISPR Cas12a based diagnostic method for peste des petits ruminants virus N gene and M gene.

Autor: Xu J; China Animal Health and Epidemiology Center, Qingdao, China., Wang Y; China Animal Health and Epidemiology Center, Qingdao, China., Zhang Y; China Animal Health and Epidemiology Center, Qingdao, China., Wang S; China Animal Health and Epidemiology Center, Qingdao, China., Su N; Qingdao Agricultural University, Qingdao, China., Chang X; China Animal Health and Epidemiology Center, Qingdao, China., Ren W; China Animal Health and Epidemiology Center, Qingdao, China., Zou Y; China Animal Health and Epidemiology Center, Qingdao, China., Liu S; China Animal Health and Epidemiology Center, Qingdao, China., Li L; China Animal Health and Epidemiology Center, Qingdao, China., Li J; China Animal Health and Epidemiology Center, Qingdao, China., Bao J; China Animal Health and Epidemiology Center, Qingdao, China., Wang Z; China Animal Health and Epidemiology Center, Qingdao, China. Electronic address: wangzhiliang@cahec.cn.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2024 Sep; Vol. 329, pp. 114971. Date of Electronic Publication: 2024 Jun 12.
DOI: 10.1016/j.jviromet.2024.114971
Abstrakt: Peste des petis ruminants (PPR) is an acute, highly contagious fatal disease affecting both domestic and wild small ruminants, caused by Morbillivirus caprinae (also known as peste des petis ruminants virus (PPRV)). Herein, a rapid method based on recombinase aided amplification-clustered regularly interspaced short palindromic repeats-Cas12a (RAA-CRISPR Cas12a) to detect PPRV was developed. CRISPR RNAs and RAA primers for PPRV-N (nucleocapsid) and PPRV-M (matrix) fragments were designed. The reaction system was constructed following screening and optimization. Detection could be completed within in 50 minutes at 37°C. Detection of gradient dilutions of plasmids carrying of PPRV N and M gene fragments indicated a minimum limit of detection of 10 copies/μL. There were no cross-reactions with related viruses and all tested lineages of PPRV were detected successfully. The method also showed good repeatability. The detection of clinical samples (previously detected using reverse transcription polymerase chain reaction (RT-PCR)) indicated good consistency between the RAA-CRISPR Cas12a method and RT-PCR. Thus, the RAA-CRISPR Cas12a method for rapid PPRV diagnosis has strong specificity, high sensitivity, and stable repeatability. Moreover, the results can be observed visually under blue or UV light or using lateral flow strips without complex instruments.
Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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