可视化等温扩增法快速筛查并分型禽流感病毒  

Visual isothermal amplification for rapid detection and genotyping of avian influenza virus

作　　者：初亚男[1] 成思佳[1] 梁超[1] 周国华[1] 

机构地区：[1]中国药科大学生命科学与技术学院,南京市210009

出　　处：《中华实用诊断与治疗杂志》2011年第7期670-673,676,共5页Journal of Chinese Practical Diagnosis and Therapy

基　　金：国家自然科学基金(20975113);江苏省科技支撑计划-社会发展基金(BE2010605)

摘　　要：目的:建立一种通用筛查禽流感病毒及其3种亚型的快速核酸检测方法。方法:对20例已知样本扩增禽流感病毒共有的M基因上的一段保守序列快速筛查并确定是否为禽流感病毒,扩增禽流感病毒(H5N1,H7N7,H9N2)3种亚型特异性HA基因上一段序列鉴定禽流感病毒的基因型。采用环介导等温扩增法检测核酸,用肉眼观察双链嵌合染料SYBR Green I荧光显色信号强度。结果:3种禽流感病毒亚型的检测灵敏度均为10拷贝/管,3组引物间无非特异性扩增。18例阴性,2例阳性,准确性及特异性均为100%。结论:建立的核酸检测方法适合现场快速检测禽流感病毒及其3种亚型。Objective To establish a rapid nuclear-acid detection of the avian influenza virus and three subtypes including H5N1,H7N7 and H9N2.Methods A conserved region of the M gene in all avian influenza viruses was amplified to detect the avian influenza virus.The subtypes of avian influenza virus were further identified by amplifying a subtype-specific sequence of HA gene.The assay was performed by a highly sensitive loop-mediated isothermal amplification（LAMP） incubated at 60 ℃ for 1 hour with three pairs of LAMP primers and Bst DNA polymerase.The visual detection was readily realized through the incorporation of SYBR Green I fluorescence to the LAMP reaction system.Results The sensitivity of detecting three subtypes was 10 DNA copies per tube,and there was no non-specific amplification among the detection of the three virus subtypes.Both of the accuracy and the specificity were 100% in 18 negative samples and 2 positive samples.Conclusion The established rapid detection is suitable for point-of-care tests of avian influenza virus and three subtypes.

关 键 词：禽流感病毒 环介导等温扩增 H5N1亚型 H7N7亚型 H9N2亚型 

分 类 号：S852.65[农业科学—基础兽医学]