猪流行性腹泻病毒实时荧光定量RT-PCR检测方法的建立

Development of Real-time Quantitative RT-PCR Method for Detecting Porcine Epidemic Diarrhea Virus

根据Gen Bank报道的N基因高度保守核苷酸序列,设计并合成一对引物。上下游引物与Gen Bank中登录的153株猪流行性腹泻病毒(PEDV)N基因全长序列匹配度分别是100%和97%。以本实验室分离流行毒株为模板,利用SYBR Green I荧光染料法进行RT-PCR扩增,获得扩增产物构建重组质粒作为阳性对照,建立检测猪流行性腹泻病毒核酸的方法。同一样品进行3次重复试验,变异系数<0.9%。通过对临床样品进行检测和测序验证,核酸检测结果中的阳性样品准确率为100%。本研究所建立的荧光定量PCR检测方法具有快速、灵敏、准确等优点,可用于临床PEDV的检测及分子流行病学调查。

A pair of primers was synthesized according to the conservative sequence of PEDV N genes that have been retrieved from Gen Bank.The forward and reverse primers were aligned with 153 full-length sequences of PEDV N genes,and exhibited identifies of 100% and 97%,respectively.The fluorescent dye SYBR Green I was used to develop the real-time RT-PCR method.The N gene of an isolated PEDV was amplified by RT-PCR,subcloned into a plasmid,and then was used as a positive control.The coefficient of variation was less than 0.9% in triplicate assays.Based on positive clinical samples,100% agreement was achieved by using this real-time RT-PCR method.This method would be useful in diagnostic analysis against PEDV in epidemiological investigations.