Development and evaluation of a one-step multiplex real-time TaqMan® RT-qPCR assay for the detection and genotyping of equine G3 and G14 rotaviruses in fecal samples

Abstract

Background Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and are significantly time-consuming, costly and labor intensive. Here, we developed the first one-step multiplex TaqMan ® real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes for the rapid detection and G-typing directly from fecal specimens. Methods A one-step multiplex TaqMan ® RT-qPCR assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes was designed. The analytical sensitivity was assessed using serial dilutions of in vitro transcribed RNA containing the target sequences while the analytical specificity was determined using RNA and DNA derived from a panel of group A rotaviruses along with other equine viruses and bacteria. The clinical performance of this multiplex assay was evaluated using a panel of 177 fecal samples and compared to a VP7-specific standard RT-PCR assay and Sanger sequencing. Limits of detection (LOD), sensitivity, specificity, and agreement were determined. Results The multiplex G3 and G14 VP7 assays demonstrated high specificity and efficiency, with perfect linearity. A 100-fold difference in their analytical sensitivity was observed when compared to the singleplex assays; however, this difference did not have an impact on the clinical performance. Clinical performance of the multiplex RT-qPCR assay demonstrated that this assay had a high sensitivity/specificity for every target (100% for NSP3, > 90% for G3 VP7 and > 99% for G14 VP7, respectively) and high overall agreement (> 98%) compared to conventional RT-PCR and sequencing. Conclusions This new multiplex RT-qPCR assay constitutes a useful, very reliable tool that could significantly aid in the rapid detection and G-typing of ERVA strains circulating in the field.

Mouse-Adapted SARS-CoV-2 MA10 Strain Displays Differential Pulmonary Tropism and Accelerated Viral Replication, Neurodissemination, and Pulmonary Host Responses in K18-hACE2 Mice

“…For absolute quantitation of viral RNA, a standard curve using a synthesized in vitro transcribed RNA containing a 132-bp fragment from the SARS-CoV-2 ORF1ab gene was used. Briefly, the target sequence was chemically synthesized and cloned into the pGEM-3Z vector (Promega, Madison, WI), and in vitro RNA transcription was performed as previously described ( 71 , 72 ). Synthesized RNA was purified using the MEGAclear transcription clean-up kit (Thermo Fisher Scientific) and quantitated using a Qubit 4 fluorometer (Invitrogen).…”

Rotaviruses: From Pathogenesis to Disease Control—A Critical Review

“…The sequencing of VP7, VP4, and other genome segments is required for genotyping circulating rotavirus strains. Conventional sequencing techniques have the disadvantages of being labor-intensive, low throughput, and costly [ 141 ]. Newer RT-qPCR assays, especially TaqMan ® assays, have been used to overcome the challenges of conventional RT-PCR and sequencing.…”

“…Newer RT-qPCR assays, especially TaqMan ® assays, have been used to overcome the challenges of conventional RT-PCR and sequencing. Compared with other methods, real-time quantitative PCR has advantages of increased specificity, sensitivity, genotyping, ability to multiplex, high throughput sample processing, faster turnaround time, and quantitative accuracy [ 141 ]. For complete characterization of RV genome and identification of unusual genotype constellations, whole genome analysis has recently been recommended by the RV classification working groups [ 20 ].…”

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

“…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].…”