Development of a Real-Time Reverse Transcription-PCR Assay To Detect and Quantify Group A Rotavirus Equine-Like G3 Strains

• Published in: Journal of clinical microbiology 2021-10, Vol.59 (11), p.e0260220-e0260220
• Main Authors: Katz, Eric M, Esona, Mathew D, Gautam, Rashi, Bowen, Michael D
• Format: Article
• Language: English
• Subjects: Animals; Feces; Genotype; Horses; Humans; Phylogeny; Real-Time Polymerase Chain Reaction; Reverse Transcription; RNA, Viral - genetics; Rotavirus - genetics; Rotavirus Infections - diagnosis; Rotavirus Infections - veterinary; Virology
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.02602-20

Since 2013, group A rotavirus strains characterized as novel DS-1-like intergenogroup reassortant "equine-like G3" strains have emerged and spread across 5 continents among human populations in at least 14 countries. Here, we report a novel one-step TaqMan quantitative real-time reverse transcription-PCR assay developed to genotype and quantify the viral load for samples containing rotavirus equine-like G3 strains. Using a universal G forward primer and a newly designed reverse primer and TaqMan probe, we developed and validated an assay with a linear dynamic range of 227 to 2.3 × 10 copies per reaction and a limit of detection of 227 copies. The percent positive agreement, percent negative agreement, and precision of our assay were 100.00%, 99.63%, and 100.00%, respectively. This assay can simultaneously detect and quantify the viral load for samples containing DS-1-like intergenogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost. It will be valuable for high-throughput screening of stool samples collected to monitor equine-like G3 strain prevalence and circulation among human populations throughout the world.