鸭黄病毒巢式PCR检测方法的建立和应用

福建农业学报 ›› 2012, Vol. ›› Issue (2): 124-129.

鸭黄病毒巢式PCR检测方法的建立和应用

张琳^1,2, 胡北侠^1,2, 颜世敢^1,2, 杨少华^1,2, 许传田^1,2, 张秀美^1,2

1. 山东省农业科学院畜牧兽医研究所, 山东济南 250100;
2. 山东省畜禽疫病防治与繁育重点实验室, 山东济南 250100

收稿日期:2011-04-02 修回日期:2011-11-19 出版日期:2012-02-29 发布日期:2012-02-29
通讯作者: 张秀美(1956-),女,研究员,主要从事家禽疫病诊断与防治技术研究
基金资助:
国家公益性行业(农业)科研专项(201003012)

Development and Application of the Nested PCR Assay for Duck Flavivirus

ZHANG Lin^1,2, HU Bei-xia^1,2, YAN Shi-gan^1,2, YANG Shao-hua^1,2, XU Chuan-tian^1,2, ZHANG Xiu-mei^1,2

1. Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Science, Jinan, Shandong 250100, China;
2. Shandong Key Lab of Animal Disease Control and Breeding, Jinan, Shandong 250100, China

Received:2011-04-02 Revised:2011-11-19 Online:2012-02-29 Published:2012-02-29

摘要/Abstract

摘要： 参照GeneBank已登录的黄病毒基因序列设计通用引物,建立基于黄病毒通用引物的巢式PCR检测体系,为黄病毒特别是新发黄病毒提供了高效的检测方法。该方法通过基因组的比对,选择NS5基因保守区设计了4条通用简并引物Flav P1~Flav P4,在此基础上建立了巢式PCR反应体系,并应用到鸭黄病毒的检测中。该体系仅能扩增鸭黄病毒目的基因而不能扩增其他病毒,且敏感性达到90 copies·μL^-1,比普通PCR高出1 000倍。同源性和进化分析表明,鸭黄病毒属蚊媒黄病毒类恩塔亚病毒群,与坦布苏病毒及近期发现的BYD病毒关系最近。以上结果表明,设计的黄病毒引物具有良好的通用性和特异性,其巢式PCR敏感性高,该体系成功地进行了鸭黄病毒的检测,验证了该方法的敏感性和特异性。

关键词: 黄病毒, 通用引物, 巢式PCR, 鸭, 敏感性

Abstract: Flavivirus universal primer pairs and nested PCR were developed as an efficient method to detect flavivirus, especially new type flavivirus. Four primers Flav P1-Flav P4 located in conserved region of the NS5 gene were designed after alignment of the flavivirus genome and then nested PCR assay was developed for duck flavivirus. Nested PCR was 1 000 times more sensitive than the conventional PCR and it was only able to amplify the flavivirus with the sensibility of 90 copies·μL^-1. Phylogenetic analysis showed that duck flavivirus belongs to mosquito-born flavivirus, NTAV group, similar with Tembusu and BYD virus. The clinical samples detection showed that the universal primers and nested PCR were highly specific and sensitive. In addition, the station of evolution of duck flavivirus was clarified.

Key words: Flavivirus, universal primers, nested PCR, duck, sensitivity

中图分类号:

S 852.65

张琳, 胡北侠, 颜世敢, 杨少华, 许传田, 张秀美. 鸭黄病毒巢式PCR检测方法的建立和应用[J]. 福建农业学报, 2012, (2): 124-129.

ZHANG Lin, HU Bei-xia, YAN Shi-gan, YANG Shao-hua, XU Chuan-tian, ZHANG Xiu-mei. Development and Application of the Nested PCR Assay for Duck Flavivirus[J]. Fujian Journal of Agricultural Sciences, 2012, (2): 124-129.

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