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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (2): 48-52.doi: 10.11924/j.issn.1000-6850.2011-1758

所属专题： 畜牧兽医

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

禽流感病毒RT-PCR ELISA诊断方法的建立

杨少华 胡北侠 许传田 颜士敢 张琳 黄艳艳 张秀美   

  • 收稿日期:2011-06-13 修回日期:2011-08-09 出版日期:2012-01-15 发布日期:2012-01-15

Establishment of a RT-PCR method for detection of Avian Influenza virus

  • Received:2011-06-13 Revised:2011-08-09 Online:2012-01-15 Published:2012-01-15

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摘要：

针对A型禽流感（AI）保守的M基因设计引物，并分别用地高辛和生物素标记，建立检测A型AIV的RT-PCR ELISA方法。试验主要对RT-PCR ELISA的各种反应条件进行了优化，确定了2 μg/mL的链霉亲和素包被、1：3000抗地高辛的过氧化物酶及孵育时间等最适反应条件。该方法的敏感性比常规琼脂糖凝胶电泳检测方法高l00倍以上，而且特异性强，克服了组织样品中AIV含量少而难以检测的困难，为禽流感的早期诊断和分子流行病学调查提供了一条新途径。

关键词: 土壤, 土壤, 重金属污染, 植物修复

Abstract:

In this study a RT-PCR ELISA method was established using the primer which was designed according to M gene sequence of AIV and labeled by biotin and DIG.. The optimal reaction conditions were determined including the streptavidin was 2?g/mL and anti-digoxigenin-POD dilution titer was 1:3000. This method had a strong specificity and high sensitivity. The sensitivity was more than 100 times compared to RT-PCR, so that it could overcome the difficulty for the little AIV in the samples．It provides a new way for the molecular epidemiologic investigation and early diagnosis of AIV