Development of Real-time PCR for Detection of Porcine Reproductive and Respiratory Syndrome Virus

HUANG Juan; JIANG Ping; ZHANG Chang-yin; TANG Tai-shan; LI Yong-dong; ZHANG Zhi-tao

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October 2005

HUANG Juan, JIANG Ping, ZHANG Chang-yin, TANG Tai-shan, LI Yong-dong and ZHANG Zhi-tao. Development of Real-time PCR for Detection of Porcine Reproductive and Respiratory Syndrome Virus[J]. Virologica Sinica, 2005, 20(5): 530-533.

Citation: HUANG Juan, JIANG Ping, ZHANG Chang-yin, TANG Tai-shan, LI Yong-dong, ZHANG Zhi-tao. Development of Real-time PCR for Detection of Porcine Reproductive and Respiratory Syndrome Virus .VIROLOGICA SINICA, 2005, 20(5) : 530-533.

猪繁殖与呼吸综合征病毒实时PCR检测方法的建立

黄娟 ¹ ,
姜平 ^1* ,
张常印 ² ,
唐泰山 ² ,
李永东 ¹ ,
张治涛 ¹

1.
1.江苏南京南京农业大学农业部动物疫病诊断与免疫重点开放实验室 2.江苏出入境检验检疫局动物检验室

摘要

设计合成了一套引物和TaqMan探针,以扩增猪繁殖与呼吸综合征病毒的核衣壳蛋白基因,通过反应条件的优化,在国内首次建立了快速定量检测猪繁殖与呼吸综合征病毒的实时PCR方法,并用该法检测患病猪的肺脏等样品。结果表明该方法具有较好的特异性和重复性,对PRRSV细胞培养物的检测下限为0.01TCID50,敏感性比常规RT-PCR高100倍;对10份PRRS疑似猪肺脏样品检测5份为阳性,与病毒分离的阳性符合率为100%。该方法具有快速、灵敏、准确、低污染等优点,在PRRSV的早期检测、预防控制、进出口检疫及基础研究中会起到重要作用。
- 猪繁殖与呼吸综合征病毒
- , GP3
- , 原核表达

Development of Real-time PCR for Detection of Porcine Reproductive and Respiratory Syndrome Virus

Abstract

Primers and probe were designed from the nucleotide sequence within the ORF7 of Porcine reproductive and respiratory syndrome virus(PRRSV),and a quantitative TaqMan real-time PCR was developed for PRRSV detection.Specificity and sensitivity were compared with virus isolation and a conventional RT-PCR,and were found to be equal or superior to the reference methods.Reproducibility was tested and proved that the assay was reliable.Standard dilutions allowed absolute quantitation of the amount of viral RNA.The TaqMan real-time PCR described below is time-saving,easy to handle,exhibits a decreased risk of cross-contamination and is highly sensitive and specific.It is considered to be a powerful tool for the rapid detection of PRRSV.
- PRRSV
- , GP3
- , Prokaryotic expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Development of Real-time PCR for Detection of Porcine Reproductive and Respiratory Syndrome Virus

Abstract: Primers and probe were designed from the nucleotide sequence within the ORF7 of Porcine reproductive and respiratory syndrome virus(PRRSV),and a quantitative TaqMan real-time PCR was developed for PRRSV detection.Specificity and sensitivity were compared with virus isolation and a conventional RT-PCR,and were found to be equal or superior to the reference methods.Reproducibility was tested and proved that the assay was reliable.Standard dilutions allowed absolute quantitation of the amount of viral RNA.The TaqMan real-time PCR described below is time-saving,easy to handle,exhibits a decreased risk of cross-contamination and is highly sensitive and specific.It is considered to be a powerful tool for the rapid detection of PRRSV.