Journal Article Epublish
PubMed: 40725091 DOI: 10.3390/ijms26146846
CRISPR/Cas12a-Based One-Tube RT-RAA Assay for PoRV Genotyping.
Int J Mol Sci 26(14)
Authors: Mingfang Bi, Zunbao Wang, Kaijie Li, Yuhe Ren, Dan Ma, Xiaobing Mo
Affiliations
- College of Veterinary Medicine, State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, and Institute of Zoonosis, Jilin University, Changchun 130062, China. CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
- College of Veterinary Medicine, State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, and Institute of Zoonosis, Jilin University, Changchun 130062, China.
Abstract
Porcine rotavirus (PoRV), a primary etiological agent of viral diarrhea in piglets, frequently co-infects with other enteric pathogens, exacerbating disease severity and causing substantial economic losses. Its genetic recombination capability enables cross-species transmission potential, posing public health risks. Globally, twelve G genotypes and thirteen P genotypes have been identified, with G9, G5, G3, and G4 emerging as predominant circulating strains. The limited cross-protective immunity between genotypes compromises vaccine efficacy, necessitating genotype surveillance to guide vaccine development. While conventional molecular assays demonstrate sensitivity, they lack rapid genotyping capacity and face technical limitations. To address this, we developed a novel diagnostic platform integrating reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR-Cas12a. This system employs universal primers for the simultaneous amplification of G4/G5/G9 genotypes in a single reaction, coupled with sequence-specific CRISPR recognition, achieving genotyping within 50 min at 37 °C with 10 0 copies/μL sensitivity. Clinical validation showed a high concordance with reverse transcription quantitative polymerase chain reaction (RT-qPCR). This advancement provides an efficient tool for rapid viral genotyping, vaccine compatibility evaluation, and optimized epidemic control strategies.
MeSH
Recombination, Genetic, Clustered Regularly Interspaced Short Palindromic Repeats, Vaccines, Reverse Transcription, Genotype