人多瘤病毒KI和WU PCR检测方法的建立及应用  

Development and application of TaqMan probe real-time PCR assay for detection of KIPyV and WUPyV

作　　者：张骞[1] 郑文芝[1] 袁武梅[1] 麻粉莲[1] 郑丽舒[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所卫生部医学病毒和病毒病重点实验室,北京100052

出　　处：《中华实验和临床病毒学杂志》2015年第3期266-269,共4页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的 针对人多瘤病毒KI株和WU株,建立一种快速、特异、灵敏的标准SOP,并初步应用于200例北京地区呼吸道感染儿童的NPA（Nasopharyngea Aspirates）的检测.方法 设计巢式PCR和实时荧光定量PCR方法,检测KIPyV和WUPyV基因,并对扩增产物进行序列分析,比较其应用于儿童NPA的检测效果.结果 KIPyV和WUPyV的TaqMan探针实时荧光定量PCR检测灵敏性分别为10拷贝/μl和1拷贝/μl,高于巢式PCR（500拷贝/μl）,两种检测方法中均无其他呼吸道病毒阳性扩增.TaqMan探针实时荧光定量PCR可重复性检测中KIPyV和WUPyV的CV值分别小于2.9％和1.95％.应用巢式PCR KIPyV和WUPyV阳性检出率为1.5％和8％,TaqMan探针实时荧光定量PCR检测检出率为12％、14％.结论 本研究建立了一种灵敏性、可重复性好、特异性高的快速核酸检测方法,其在临床上有较好的应用前景。Objective We develop a rapid,specific,sensitive tandardized SOP.And initial application for 200 nasopharyngeal aspirates of children with related pathogens of acute respiratory tract infections in BeiJing area.Methods To developed nested PCR and TaqMan probe real-time fluorescence quantitative PCR method for detection of KIPyV and WUPyV＇S gene,and then the sequences of gene fragments are analyzed.Evalution of two assays from 200 nasopharyngeal aspirates.Results In this study,sensitivity of TaqMan probe real time fluorescent quantitative PCR assay was higher than one of nested PCR （500 copies/μl）,and both assays did not show any positive amplification in detetion of other respiratory virus.Coefficient of varience of KIPyV and WUPyV are less than 2.9% and 1.95% respectively in the repeatability detection.The detection rates of KIPyV,and WUPyV were 1.5% and 8% in nested PCR assay and 12% and 14% in real time Fluorescent quantitative PCR assay respectively.Conclusion This study established good sensitivity and reproducibility,high specificity and rapid method for detection nucleic acid of these polyomaviruses that have good prospects on the clinical application.

关 键 词：多病病毒属 逆转录聚合酶链反应 

分 类 号：R440[医药卫生—诊断学]