作 者：
郭捷;谢芝勋;罗思思;刘加波;邓显文;谢志勤;庞耀珊;范晴
关键词：
H1亚型AIV;SYBR GreenⅠ;实时荧光定量PCR
摘 要：
本研究旨在建立一种检测H1亚型禽流感病毒(AIV)的SYBR GreenⅠ实时荧光定量PCR方法。根据对GenBank中H1亚型AIV HA基因序列的分析,设计针对H1亚型AIV的HA基因特异性引物,并以H1亚型AIV的cDNA为模版,构建重组阳性质粒。采用梯度稀释重组标准品阳性质粒DNA的方法,建立SYBR GreenⅠ实时荧光定量PCR(real-time PCR)标准曲线。敏感性试验结果显示,扩增效率和标准误差分别为100.9%和0.011 7,熔解曲线呈单一熔解峰。在35个Ct值内该方法可检测到100个拷贝的标准品DNA;特异性试验结果显示,该方法对于其他亚型AIV和禽呼吸道病原体不能检出;重复性试验结果显示,组内变异系数小于1%。建立的Real-time PCR具有特异、敏感、快速、重复性好等优点,可用于临床上H1亚型AIV的检测。
译 名：
Development of Real-time Fluorescent Quantitative PCR Assay for Detection of Avian Influenza Virus H1 Subtype
作 者：
GUO Jie 1,XIE Zhi-xun 2,LUO Si-si 2,LIU Jia-bo 2,DENG Xian-wen 2,XIE Zhi-qin 2,PANG Yao-shan 2,FAN Qing 2(1.College of Animal Science and Technology,Guangxi University,Nanning,Guangxi,530004,China;2.Guangxi Veterinary Research Institute,Guangxi Key Laboratory of Animal Vaccines and New Technology,Nanning,Guangxi,530001,China)
关键词：
H1subtype of AIV;SYBR greenⅠ;Real-time PCR
摘 要：
This study was to establish a SYBR GreenⅠReal-time PCR assay for detection of H1subtype avian influenza virus.A pair of specific primers were designed and synthesized according to the sequences of H1subtype avian influenza virus(AIV)-HA in GenBank.And recombinant plasmids containing the target gene were constructed and used as the Real-time PCR standard templates.The sensitivity result showed that the gene’s melting curve had a single peak,and the amplification efficiency was 100.9%,and the stangard error was 0.011 7;also the developed SYBR GreenⅠReal-time PCR could detect 1×102 template copies of plasmid DNA in 35Ct and the gene’s coefficient of variation less than percent 1for intra-assay.The specificity as all negative controls and other avian pathogens,such as other subtypes of AIV and bird respiratory pathogens showed negative results.As a result of the study showed that the Real-time PCR will be useful for the clinical diagnosis of H1subtype avian influenza virus infection with sensitive,specific,rapid and repeatabile characteristics.
