4种呼吸道病毒悬液芯片检测方法的建立  

Establishment of a multiplex liquid bead array for simultaneous detection of four respiratory viruses

作　　者：陈沁[1,2] 裘敏燕 付永锋[3] 徐锦[4] 居丽雯[3] 程训佳[3] 李瑶[1] 

机构地区：[1]复旦大学生命科学学院,上海200433 [2]联合基因生物医药有限公司,上海200433 [3]复旦大学上海医学院,上海200032 [4]复旦大学附属儿科医院儿科研究所,上海201102

出　　处：《中国病原生物学杂志》2013年第2期97-101,130,共6页Journal of Pathogen Biology

基　　金：国家"艾滋病和病毒性肝炎等重大传染病防治"科技重大专项课题(No.2009ZX10004-104);上海市科学技术委员会上海市科技人才计划项目(No.12XD1422000)

摘　　要：目的建立一种基于悬液芯片的呼吸道病毒多重检测方法,对4种呼吸道病毒或亚型进行快速检测和鉴定,包括人类偏肺病毒,呼吸道合胞病毒,流感病毒A和流感病毒B。方法依据NCBI公开数据库上各病毒的基因序列信息,设计并合成相关引物探针序列。抽提病毒RNA,经反转录后对目的序列进行PCR扩增,产物与核酸探针微球组杂交后于BioPlex 200检测荧光信号值。结果优化了多重PCR方法,hMPV、RSV与IfB的悬液芯片检测灵敏度约3DNA拷贝,IfA的悬液芯片检测灵敏度约30DNA拷贝。4种病毒或亚型同时检测的特异性高,省时,无交叉反应。17个经传统方法鉴定为呼吸道病毒的临床标本用悬液芯片检测16个阳性,检出率94.1%。标本经测序分析验证,结果与悬液芯片法一致。结论建立的同时检测4种呼吸道病毒或亚型的悬液芯片方法特异性高,为快速筛查和鉴定呼吸道病毒提供了新的手段。Objective To establish a method for simultaneous detection of multiple respiratory viruses based on a liquid bead array in order to rapidly detect and identify four respiratory viruses or subtypes： human metapneumovirus, respirato-ry syncytial virus, influenza virus type A, and influenza virus type B. Methods Primers and probes were designed and synthesized according to genomic sequences in the NCBI GenBank database. Viral RNA was extracted and reverse-tran- scribed and target sequences were amplified. PCR products were hybridized with coupled nucleic acid probe beads set and detected by the Bio-PlexTM 200 system. Results Multiplex PCR conditions were optimized, and the limits of detection for each virus or subtype were about 3 DNA copies for hMPV, RSV, and IfB and 30 DNA copies for IrA. Simultaneously detecting the four viruses or subtypes was highly specific and saved time, and no cross-reactivity was observed. Viruses or subtypes in 17 clinical samples identified by traditional methods were tested using the established assay, and 16 of the 17 samples were confirmed to be positive. The detection rate was about 94. 1%. The results of sample sequencing matched those of the liquid bead array assay. Conclusion A multiplex liquid bead array is a highly specific way to sim-ultaneously detect four respiratory viruses or subtypes, and this assay may play a new and important role in rapid screen-ing and identification of respiratory viruses.

关 键 词：呼吸道病毒 悬液芯片 多重PCR 

分 类 号：R373.1[医药卫生—病原生物学]