【作者】 徐敏； 李志强； 黄元； 陈晶； 陈少勤； 张健騑； 赵亚华； 宣华； 向华；

【Author】 Min Xu1,ZhiQiang Li2,Yuan Huang1,Jing Chen1,Shaoqin Chen1,Jianfei Zhang1,Yahua Zhao2,Hua Xuan1,Hua Xiang1(1Veterinary Public Health Public Laboratory of Guangdong,Veterinary Medicine Institute,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;2 College of Life Science,South China Agricultural University,Guangzhou510642,China)

【机构】 广东省农业科学院兽医研究所广东省兽医公共卫生公共实验室； 华南农业大学生命科学学院；

【摘要】 为了建立一种在猪流感流行病学调查和临床诊断中应用的快速高效的诊断方法(目的),根据猪流感病毒(Swine Influenza virus,SIV)M基因的保守序列,设计并合成一对特异性引物和一条特异性TaqMan探针,建立了以RNA为反应模板的一步法荧光定量RT-PCR诊断方法(方法)。使用含有M基因的重组质粒作为标准品绘制标准曲线,该曲线效率值为2.014,误差值为0.005。实验结果表明,该方法灵敏度极高,最低可检测到10个拷贝的核酸,临床样品检测结果也显示其灵敏度高于普通PCR和病毒分离法;特异性强,对猪群常见的其他6种病毒检测结果均为阴性(结果)。临床样品检测显示该方法操作简单,可以用RNA作为模板直接检测,节省时间,而且特异性好灵敏度高,是可以应用于猪流感流行病学调查的一种可靠的诊断方法(结论)。更多还原

【Abstract】 In order to develop a rapid and high-performance assay for epidemiological study and clinical diagnosis of swine influenza virus,a one-step real-time quantitative RT-PCR assay based on a TaqMan probe was developed for detection of swine influenza viruses RNA.Conserved regions in the M gene of swine influenza viruses served as targets for primers and TaqMan probe.Concentrations of primers and probe were optimized to improve the sensitivity and specificity of the reactions.A series of dilutions of recombinant plasmid were prepared and used to generate standard curve with a efficiency value of 2.014 and a error value of 0.005.The threshold of 10 copies of target molecules could be detected revealed that the method was very sensitive and the results of the clinical diagnosis showed that this assay was more sensitive than the normal RT-PCR assay and the virus isolation.It was negative for other six popular viruses in swine showed that the assay was highly specify.This assay could be used for rapid detection and monitoring in the epidemiology study of SIV,because it was easy to operate and very timesaving.更多还原