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
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
复制标题
DOI：
10.1186/s12985-019-1149-1
复制
发表时间：
2019-04-25
期刊：
影响因子：
4.8
通讯作者：
Balasuriya, Udeni B. R.
中科院分区：
文献类型：
--
作者：
Carossino, Mariano;Barrandeguy, Maria E.;Balasuriya, Udeni B. R.
背景马轮状病毒A(Erva)是引起新生马驹腹泻的主要原因，对世界各地的马匹养殖企业都有负面影响。在感染马驹的Erva菌株中，G3P[12]和G14P[12]基因型最常见，而具有其他基因组排列的菌株感染较少。埃博拉病毒流行株的鉴定对于诊断和监测以及了解其分子流行病学至关重要。目前可用于影响其他动物物种的Erva和轮状病毒的基因分型方法依赖于Sanger测序，而且非常耗时、昂贵和劳动密集型。为此，我们首次建立了针对Erva G3和G14基因NSP3和VP7的一步多重实时荧光定量RT-qPCR方法，用于快速检测和直接从粪便标本中进行G分型。用含有目标序列的体外转录RNA的一系列稀释度来评估分析的敏感性，而分析的特异性则用来自A组轮状病毒以及其他马病毒和细菌的RNA和DNA来确定。使用177份粪便样本评估了这种多重检测的临床性能，并与VP7特异的标准RT-PCR检测和Sanger测序进行了比较。结果G3和G14 VP7多重检测具有较高的特异性和高效性，具有良好的线性关系。与单链分析相比，它们的分析灵敏度有100倍的差异；然而，这种差异对临床表现没有影响。结论与常规RT-PCR和测序方法相比，该方法对所有靶点均有较高的灵敏度/特异度(NSP3为100%，G3 VP7为90%，G14 VP7为99%)，总体符合率为98%。结论该方法是一种实用、可靠的检测方法，可用于现场快速检测和G分型。
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((R)) 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((R)) 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.