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

Mariano Carossino,Udeni B R Balasuriya,Yanqiu Li,Erdal Erol,Maria E Barrandeguy

doi:10.1186/s12985-019-1149-1

Mariano Carossino, Udeni B R Balasuriya + Show 3 more

Open Access

https://doi.org/10.1186/s12985-019-1149-1

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Abstract

BackgroundEquine 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.MethodsA 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.ResultsThe 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.ConclusionsThis 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.

Highlights

Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide
Analysis of fecal samples by VP7-specific RT-PCR and sequencing for determination of G-types A total of 177 fecal samples were included in the study, from which 92 samples were confirmed negative for ERVA, while 85 were positive as determined by VP7-specific standard RT-PCR [29, 30]
Analytical sensitivity and specificity of ERVA-specific singleplex and multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays targeting G3 VP7, G14 VP7 and NSP3 genes Analytical sensitivity of ERVA-specific singleplex RT-qPCR assays The analytical sensitivity of the ERVA-specific singleplex and multiplex RT-qPCR assays was determined using a ten-fold dilution series (3 replicates per dilution) of in vitro transcribed (IVT) ribonucleic acid (RNA) (107 to 0.1 IVT RNA copies/μl) containing the target sequences

Summary

Introduction

Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Equine rotavirus A (ERVA) has been identified as the leading cause of diarrhea in neonatal foals < 3 months of age and is responsible for 20 to 77% of foal diarrhea cases, causing significant economic losses to the equine breeding enterprises [1,2,3,4,5,6,7]. The ERVA genome consists of 11 double-stranded RNA segments that encode for six structural proteins (VP1–4, 6 and 7) and six non-structural proteins (NSP1–6). Based on VP6 identity, rotaviruses are classified into eight groups (A-H), from which group A rotaviruses (RVA) are the leading cause of diarrhea in humans and several animal species, including horses [15]. Group A rotaviruses are further classified into G-types and P-types according to the nucleotide sequence of the two outer capsid proteins, VP7 and VP4, encoded by segments 9 and 4 of the genome, respectively [16]. Other genomic arrangements involving G- and P-types different from G3/G14 and P[12] have been infrequently described as infecting horses [1]

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