Quadruplex Real-Time TaqMan® RT-qPCR Assay for Differentiation of Equine Group A and B Rotaviruses and Identification of Group A G3 and G14 Genotypes

Abstract

Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan® RT-qPCR assay for differentiation of ERVA and ERVB and simultaneous G-typing of ERVA strains, evaluated its analytical and clinical performance, and compared it to (1) a previously established ERVA triplex RT-qPCR assay and (2) standard RT-PCR assay and Sanger sequencing of PCR products. This quadruplex RT-qPCR assay demonstrated high sensitivity (>90%)/specificity (100%) for every target and high overall agreement (>96%). Comparison between the triplex and quadruplex assays revealed only a slightly higher sensitivity for the ERVA NSP3 target using the triplex format (p-value 0.008) while no significant differences were detected for other targets. This quadruplex RT-qPCR assay will significantly enhance rapid surveillance of both ERVA and ERVB circulating and emerging strains with potential for interspecies transmission.

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex

“…Multiplex one-step qPCR or RT-qPCR assays allow the rapid identification of up to four targets in a single reaction. Therefore, this technique is now widely adopted in veterinary diagnostic laboratories [ 51 , 52 , 53 , 54 ]. Panels of qPCR/RT-qPCR and multiplex qPCR/RT-qPCR were previously developed for the detection of CIRDC-associated pathogens but they neither include all of the CIRDC-associated pathogens nor do they incorporate SARS-CoV-2 for its simultaneous detection [ 4 , 8 , 21 ].…”

“…Limit of detection with 95% confidence (LOD 95% ) of each assay was determined by statistical probit analysis (non-linear regression model) using SPSS 14.0 software (SPSS Inc., Chicago, IL, USA) from twelve replicates per dilution ranging from 10 3 to 10 0 copies/μL. Cycle threshold (Ct) cut-off values were determined using the following formula: Ct cut-off = Average Ct values of 12 replicates of the endpoint dilution + (3 × standard deviation [SD]) [ 53 , 54 , 65 ]. Intra-run imprecision was determined by performing 12 replicates of plasmid DNA/ IVT RNA containing 10 5 to 10 3 copies/μL on the same run and inter-run imprecision was determined by using three replicates of plasmid DNA/ IVT RNA containing 10 5 to and 10 3 copies/μl on two independent runs.…”

“…One of the limitations of multiplexing is related to the overlapping emission and spectra that some florescent dyes have, which limits their use in multiplex scenarios. This issue has been overcome in recent years with a new generation of fluorescent dyes by several manufacturers [51][52][53][54].…”

“…This issue has been overcome in recent years with a new generation of fluorescent dyes by several manufacturers. [ 51 , 52 , 53 , 54 ].…”

Equine Rotavirus A under the One Health Lens: Potential Impacts on Public Health

“…While initially characterized via serotyping and, subsequently, via genotyping of few genomic segments (i.e., that encoding for VP7 and VP4, determining G- and P-types), the emergence of NGS technologies has made surveillance and molecular epidemiology studies much more comprehensive, allowing for evaluation and comparison of full genome constellations of RVA strains from all around the globe. More rapid genotyping tools based on multiplex real-time RT-PCR have been developed and have become available for human RVA and equine RVA [ 252 , 253 , 254 , 255 , 256 , 257 , 258 ] but not for other animal RVA. These tools can be useful for rapid genotypification and further development for other animal species and are desired to contribute to monitoring.…”