A组轮状病毒原位捕获RT-qPCR检测体系的建立及评估

Development and evaluation of in situ capture RT-qPCR for detection of group A rotavirus

目的建立基于原位捕获反转录实时定量PCR方法(in situ capture real time quantitative reverse transcription PCR,ISC-RT-qPCR)检测感染性A组轮状病毒(group A rotavirus,RVA)的体系,并对其进行评估。方法以自行构建的NSP3重组质粒为对象,绘制标准曲线;通过优化ISC-RT-qPCR体系中猪胃粘膜提取物(porcine gastric mucin,PGM)包被浓度、孵育时间、孵育缓冲液pH和封闭时间的参数,建立、完善RVA的ISC-RT-qPCR检测体系,并评估该体系的特异性、检测限及稳定性。结果ISC-RT-qPCR最佳试验参数分别为:PGM包被浓度为1.0 mg/mL、孵育时间为60 min、孵育pH为9.0、封闭时间为60 min;体系的检测限约为5拷贝/mL,检测特异性强、稳定性好。结论本研究所建立的ISC-RT-qPCR是一种可用于检测感染性RVA的方法,具有操作简单、绿色环保、可高通量检测等优点,适合于临床、尤其是低病毒载量的食品和环境样品中RVA的快速检测。

Objective To develop and evaluate the in situ capture real time quantitative reverse transcription PCR(ISC-RT-qPCR)method for the detection of infectious group A rotavirus(RVA).Methods The standard curve for the assay was plotted with a self-constructed NSP3 recombinant plasmid.The ISC-RT-qPCR for the detection of RVA was established and optimized.The optimized parameters included coating concentration of porcine gastric mucin(PGM),incubation time,pH of incubation buffer and blocking time.Besides,the specificity,detection limit and stability of the system were evaluated.Results The optimal test parameters of ISC-RT-qPCR were:PGM coating concentration of 1.0 mg/mL,incubation of samples for 60 min at pH 9.0 and blocking time of 60 min.Under the optimal test conditions,the detection limit of ISC-RT-qPCR was about 5 copies/mL with high specificity,accuracy and stability.Conclusions The ISC-RT-qPCR established in this study is a simple,environmentally friendly and high-throughput method to detect the infective RVA and is suitable for rapid detection of low-load RVA in food and environmental samples.