Development of Fluorescent Quantitative PCR Method to Rapidly Detect Pseudorabies Virus

WAN Chao; WEN Guo-yuan; PAN Zi-shu; ZHANG Chu-yu; XIONG Zhong-liang; YANG Jun; AI Di-yun

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October 2006

WAN Chao, WEN Guo-yuan, PAN Zi-shu, ZHANG Chu-yu, XIONG Zhong-liang, YANG Jun and AI Di-yun. Development of Fluorescent Quantitative PCR Method to Rapidly Detect Pseudorabies Virus[J]. Virologica Sinica, 2006, 21(5): 485-489.

Citation: WAN Chao, WEN Guo-yuan, PAN Zi-shu, ZHANG Chu-yu, XIONG Zhong-liang, YANG Jun, AI Di-yun. Development of Fluorescent Quantitative PCR Method to Rapidly Detect Pseudorabies Virus .VIROLOGICA SINICA, 2006, 21(5) : 485-489.

快速检测伪狂犬病毒荧光定量PCR技术建立及应用

万超 ¹ ,
温国元 ¹ ,
潘兹书 ^1* ,
张楚瑜 ¹ ,
熊中良 ² ,
杨峻 ² ,
艾地云 ²

1.
1.武汉大学生命科学学院，病毒学国家重点实验室，湖北武汉 430072
2.
湖北省农业科学院畜牧兽医研究所，湖北武汉 430209

摘要

以伪狂犬病毒(PRV)保守的gE基因序列为参考，设计、优化出一对特异的PCR引物和一条TaqMan荧光探针，结合RotorGene检测系统，建立一种快速定量检测伪狂犬病毒的荧光定量PCR技术。该方法线形范围为1.0×102-1.0×107拷贝/L，灵敏度达102拷贝/LDNA，比常规PCR高10倍。检测的特异性明显高于常规PCR，同时避免了常规PCR因电泳造成的污染。应用该技术检测66例猪组织或鼻咽拭子样品，阳性42份，阳性检出率为63.6％(42/66)。与病毒分离培养、常规PCR相比较结果显示，该方法具有快速、灵敏、特异、重复性好和能定量检测等优点，该方法可用于猪场PRV感染的快速定量检测和肉类食品进出口检疫。
- 伪狂犬病毒
- , 荧光定量PCR
- , 定量检测

Development of Fluorescent Quantitative PCR Method to Rapidly Detect Pseudorabies Virus

1.
1.College of Life Sciences, State Key Laboratory of Virology, Wuhan University, Wuhan 430072, China
2.
Institute of Animal Husbandry and Veterinary Science, Hubei Academy of Agricultural Science, Wuhan 430209 , China

Abstract

A fluorescent quantitative PCR (FQ-PCR) method based on sequences of the conserved gE of the PRV genome was established and evaluated after selection of optimal reaction conditions. A pair of primers and a fluorescent TaqMan probe specific for gE gene were designed and used. We compared the FQ-PCR assay to conventional virus culture techniques and PCR test. According to our results, the dynamic range of the FQ-PCR assay is between 1.0×102 copies/L and 1.0×107copies/L, and the lowest DNA concentration of detection is 1.0×102 copies/L, which is 10 fold better than regular PCR and also avoids non-specific amplification occasionally seen in regular PCR. We tested 66 specimens from different pig farms in Hubei, Henan and Gansu in 2005 by the FQ-PCR assay and isolates of PRV were found in 42 samples (63.6%). In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate and can be used to rapidly detect wild type PRV from pig’s tissues and respirator specimens.
- Pseudorabies virus (PRV)
- , Fluorescent quantitative PCR
- , Quantitative detection

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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 Fluorescent Quantitative PCR Method to Rapidly Detect Pseudorabies Virus

1. 1.College of Life Sciences, State Key Laboratory of Virology, Wuhan University, Wuhan 430072, China
2. Institute of Animal Husbandry and Veterinary Science, Hubei Academy of Agricultural Science, Wuhan 430209 , China

Abstract: A fluorescent quantitative PCR (FQ-PCR) method based on sequences of the conserved gE of the PRV genome was established and evaluated after selection of optimal reaction conditions. A pair of primers and a fluorescent TaqMan probe specific for gE gene were designed and used. We compared the FQ-PCR assay to conventional virus culture techniques and PCR test. According to our results, the dynamic range of the FQ-PCR assay is between 1.0×102 copies/L and 1.0×107copies/L, and the lowest DNA concentration of detection is 1.0×102 copies/L, which is 10 fold better than regular PCR and also avoids non-specific amplification occasionally seen in regular PCR. We tested 66 specimens from different pig farms in Hubei, Henan and Gansu in 2005 by the FQ-PCR assay and isolates of PRV were found in 42 samples (63.6%). In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate and can be used to rapidly detect wild type PRV from pig’s tissues and respirator specimens.