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当前位置: 首页 > 文章 > 猪流行性腹泻病毒实时荧光定量RT-PCR检测方法的建立 中国动物检疫 2016 (1) 62-66
Position: Home > Articles > Development of Real-time Quantitative RT-PCR Method for Detecting Porcine Epidemic Diarrhea Virus China Animal Health Inspection 2016 (1) 62-66

猪流行性腹泻病毒实时荧光定量RT-PCR检测方法的建立

作  者：
劳秀杰;王静静;郑东霞;邵春艳;何海建;王晓洁;王志亮;王晓杜;宋厚辉
单  位：
浙江农林大学动物科技学院;中国动物卫生与流行病学中心;金华职业技术学院农业与生物工程学院
关键词：
猪流行性腹泻病毒;N基因;实时荧光定量RT-PCR;检测
摘  要：
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。
译  名：
Development of Real-time Quantitative RT-PCR Method for Detecting Porcine Epidemic Diarrhea Virus
作  者：
Lao Xiujie;Wang Jingjing;Zheng Dongxia;Shao Chunyan;He Haijian;Wang Xiaojie;Wang Zhiliang;Wang Xiaodu;Song Houhui;College of Animal Science and Technology,Zhejiang A&F University;China Animal Health and Epidemiology Center;School of Agricultural and Biological Engineering,JinHua Polytechnic;
关键词：
porcine epidemic diarrhea virus;;N gene;;real-time fluorescent quantitative RT-PCR;;test
摘  要：
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.

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