鉴别猪伪狂犬病病毒gE基因缺失疫苗和野毒感染的二重PCR诊断方法的建立和应用

doi:10.16431/j.cnki.1671-7236.2015.04.014

›› 2015, Vol. 42 ›› Issue (4): 865-870.doi: 10.16431/j.cnki.1671-7236.2015.04.014

鉴别猪伪狂犬病病毒gE基因缺失疫苗和野毒感染的二重PCR诊断方法的建立和应用

赵雪丽¹, 闫若潜¹, 吴志明¹, 曹伟伟¹, 王淑娟¹, 谢彩华¹, 马震原¹, 周兵强², 王东方¹

1. 河南省动物疫病预防控制中心, 郑州 450008;
2. 河南科技大学动物科技学院, 洛阳 471003

收稿日期:2014-09-25 出版日期:2015-04-20 发布日期:2015-05-05
通讯作者: 吴志明 E-mail:wuzhiming6@sina.com
作者简介:赵雪丽(1979-),女,河南太康人,博士,高级兽医师,研究方向:动物疫病病原学诊断技术研究,E-mail:zhaoxueli2002@163.com
基金资助:
河南省科技攻关计划项目“猪流行性腹泻病毒分子流行病学调查及其致病性研究”(132102110159)

Establishment and Application of a Duplex PCR Assay for Detection of Pseudorabies Virus (PRV) gE-deleted Vaccine and PRV

ZHAO Xue-li¹, YAN Ruo-qian¹, WU Zhi-ming¹, CAO Wei-wei¹, WANG Shu-juan¹, XIE Cai-hua¹, MA Zhen-yuan¹, ZHOU Bing-qiang², WANG Dong-fang¹

1. Henan Centre for Animal Diseases Control & Prevention, Zhengzhou 450008, China;
2. College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China

Received:2014-09-25 Online:2015-04-20 Published:2015-05-05

摘要/Abstract

摘要： 为建立猪伪狂犬病病毒(PRV)gE基因缺失疫苗和野毒感染的快速鉴别诊断方法,本研究根据猪伪狂犬病野毒具有gD表面抗原基因和gE毒力基因,而基因缺失疫苗只有gD基因无gE基因的特性,针对gD/gE基因的5'端核苷酸序列自行设计引物,建立鉴别PRV gE基因缺失疫苗和野毒感染的二重PCR诊断方法,并进行了特异性、敏感性和重复性试验;利用所建立的检测方法对临床疑似样品进行了检测.结果表明,成功建立了鉴别PRV gE基因缺失疫苗和野毒感染的二重PCR诊断方法,该方法灵敏度高,最低检出限为100拷贝/μL;重复性好;特异性强,可特异性地扩增出PRV细胞毒中的gD和gE基因及gE基因缺失疫苗毒中的gD基因,但对PK-15细胞和猪流行性腹泻病毒等其他8种病原扩增不出任何条带;自835份临床疑似PRV感染病料中共检测出PRV gD和gE基因双阳性样品即野毒感染阳性样品267份,PRV gD基因单阳性样品28份,选取该方法检测出的26份PRV野毒感染阳性样品用于病原分离培养,两种方法的符合率为96.1%.说明本试验建立的二重PCR鉴别诊断方法快速、灵敏、特异,对于临床上疫苗毒和野毒感染的快速鉴别诊断具有重要意义.

关键词: 猪伪狂犬病病毒; gE基因缺失疫苗; gD/gE基因; 二重PCR; 鉴别诊断

Abstract: A duplex PCR assay for detection of pseudorabies virus (PRV) gE-deleted vaccine and PRV was established using the primers designed based on the characteristics of PRV possessing surface antigen gD and gE virulence genes, and PRV gE-deleted vaccine only owing gD gene but no gE gene. The specificity, sensitivity and repetition tests were conducted, moreover, samples which were taken from clinic suspicious PRV infected pigs had been testified by the established duplex PCR assay. The results showed that a duplex PCR assay for detection of PRV gE-deleted vaccine and PRV was successfully established. The sensitivity and specificity of the duplex PCR assay revealed that the threshold of the duplex PCR was 100 copies/μL of gD or gE gene, and gD/gE gene could be amplified specifically from wild virus or gD gene from PRV gE-deleted vaccine but no products were amplified from the nucleic acid of PK-15 cell or the other 8 kinds of pathogenic viral or bacterial microorganism. The four repetition tests indicated that the duplex PCR was repeatable. Total of 267 gD and gE positive samples and 28 gD positive samples were detected from 835 clinic suspicious PRV infected pigs. Selecting 26 PRV gD and gE positive samples detected by the duplex PCR assay for pathogen cell culture, the coincidence rate between the two methods was 96.1%. The study suggested that the established duplex PCR method was highly specific and sensitive, and was suitable for clinic rapid differential diagnosis of PRV gE-deleted vaccine and PRV.

Key words: pseudorabies virus (PRV); gE-deleted vaccine; gD/gE gene; duplex PCR; differential diagnosis

中图分类号:

S858.28

赵雪丽, 闫若潜, 吴志明, 曹伟伟, 王淑娟, 谢彩华, 马震原, 周兵强, 王东方. 鉴别猪伪狂犬病病毒gE基因缺失疫苗和野毒感染的二重PCR诊断方法的建立和应用[J]. , 2015, 42(4): 865-870.

ZHAO Xue-li, YAN Ruo-qian, WU Zhi-ming, CAO Wei-wei, WANG Shu-juan, XIE Cai-hua, MA Zhen-yuan, ZHOU Bing-qiang, WANG Dong-fang. Establishment and Application of a Duplex PCR Assay for Detection of Pseudorabies Virus (PRV) gE-deleted Vaccine and PRV[J]. , 2015, 42(4): 865-870.

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鉴别猪伪狂犬病病毒gE基因缺失疫苗和野毒感染的二重PCR诊断方法的建立和应用

Establishment and Application of a Duplex PCR Assay for Detection of Pseudorabies Virus (PRV) gE-deleted Vaccine and PRV

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