摘要
参考H1-H15亚型禽流感病毒(AIV)的核蛋白(NP)基因序列以及H5、H6、H9亚型AIV血凝素(HA)基因,共设计4对引物,建立了2对引物扩增AIV的NP、HA基因的多重RT-PCR方法。应用此法分别对H5、H6、H9亚型AIV尿囊液RT-PCR,电泳,回收预期片段进行测序,并同Genebank的注册序列进行同源性分析。也对NDVI、BVI、BDV尿囊液以及3种亚型AIV尿囊液的2倍系列稀释样品进行检测。结果表明,3种亚型AIV尿囊液均出现2条特异性条带,经测序确证为目的扩增片段,BLAST的同源性初步判断3种AIV的亚型为H5N1、H6N2、H9N20对NDVI、BVI、BDV的RNA扩增未见2条目的条带,判断为阴性,说明该法特异性强;能对3种亚型AIV尿囊液64～128倍稀释的样品检测出AIV,表明其灵敏度高。该检测法检测的整个过程仅需4h,实现了AIV的快速检测。
Four primers were designed according to the sequences of nucleoprotein （NP） genes of all subtype avian influenza virus （AIV） and hemagglutinin （HA） genes of H5,H6,H9 subtype AIV.A multi-reverse transcription polymerase chain reaction （RT-PCR） was developed for detection of the RNA from AIV chorioallamoic fluid. Virus RNA specimens were detected with this method, which extracted from H5,H6,H9 subtype AIV, newcastle disease virus（NDV）, infectious bronchitis virus（IBV）, infectious bursal disease virus（IBDV） and series of two-times diluted samples of AIV （H5,H6,H9 subtype）. The results suggested that the amplification products for the three subtype AIV were specific, and the diagnosis were negative for NDV,IBV,IBDV. Also from the 64-128 times diluted samples AIV could he deteeted. And it approximately took 4 hours to detect one specimen. These showed that this assay was rapid, sensitive and specific.
出处
《湖北农业科学》
北大核心
2006年第1期20-23,共4页
Hubei Agricultural Sciences
基金
湖北省科技攻关项目(2004AA202B03)
