细胞培养结合实时荧光RT-PCR法快速检测7型腺病毒感染  

Determination of Adenovirus Type 7 by Cells Culture Combined with RT-PCR

作　　者：尚定昆 申宝玲[2] 郑晓群[3] 彭颖[2] 林新新[2] 

机构地区：[1]乐清市第二人民医院检验科,浙江乐清325608 [2]温州医学院检验医学院、生命科学院,浙江温州325027 [3]温州医学院附属第二医院检验科,浙江温州325027

出　　处：《中华医院感染学杂志》2008年第4期594-597,共4页Chinese Journal of Nosocomiology

基　　金：温州市科技发展计划基金项目(Y2006A033);浙江省自然科学基金项目(Y205188)

摘　　要：目的建立一种细胞培养与实时荧光RT-PCR相结合的检测7型腺病毒(ADV7)感染的技术。方法A549细胞分离培养鼻咽分泌物中的腺病毒纯化后,以感染复数(MOI)为0.1、0.5、5.0和10.0的纯化病毒感染A549细胞,分别在感染后3、6、12和24h提取总RNA进行ADV7E1A基因的实时荧光RT-PCR检测并测序验证;最后用该法直接检测鼻咽分泌物标本中的7型腺病毒。结果实时荧光RT-PCR最快可在感染后3h检测出0.5MOI病毒感染的早期转录物;感染后6h可检测出0.1MOI病毒感染的早期转录物;鼻咽分泌物标本直接感染后6h即可检测到病毒的早期转录物。结论细胞培养结合实时荧光RT-PCR法能快速、灵敏、特异地检测出感染性的7型腺病毒,具有一定的推广应用价值。OBJECTIVE To establish a method for detecting adenovirus type 7 by cells culture combined with real-time fluorescent RT-PCR.METHODS After purified adenovirus was dissociated from nasopharyngeal secretion in A549 cells,ADV7 E1A genes were detected by real-time RT-PCR assay and sequence analysis of cells infected with 0.1,0.5,5.0 and 10.0 MOI ADV7 at 3,6,12 and 24 h postinfection.Then the adenovirus in nasopharyngeal secretion was detected with the similar method.RESULTS Early transcription of E1A genes of adenovirus type 7 could be detected by real-time RT-PCR at 3 h postinfection with 0.5MOI virus;or at 6 h postinfection with 0.1MOI virus;Early transcription of E1A genes could be detected at 6 h postinfection in nasopharyngeal secretion.CONCLUSIONS The method by cells culture combined with real-time fluorescent RT-PCR is sensitive,specific and rapid.It can be applied in clinics for diagnosis of adenovirus type 7 infection.

关 键 词：腺病毒 细胞培养 实时 RT-PCR 

分 类 号：R373[医药卫生—病原生物学]