5种虫媒病毒悬浮芯片检测方法的建立  

Establishment of multiplex suspension array for simultaneous detection of five species of arboviruses

作　　者：罗渊[1] 刘伯华[1] 杨保安[1] 张永国[1] 李靖[1] 战大伟[1] 夏玉坤[1] 祝庆余[1] 

机构地区：[1]微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《解放军医学杂志》2008年第6期708-711,共4页Medical Journal of Chinese People's Liberation Army

摘　　要：目的建立同时检测1-4型登革病毒、乙型脑炎病毒、西尼罗病毒、森林脑炎病毒和黄热病病毒等5种虫媒病毒的悬浮芯片检测方法。方法根据GenBank发表的上述病毒的序列,通过生物信息学分析和参考相关文献,在黄病毒属高度保守的NS5区设计属通用引物和种特异检测探针。将探针共价交联到不同的Luminex色标微球上,利用属通用引物进行RT-PCR扩增,与混合微球杂交,最后利用Luminex200仪器分析荧光强度值。结果将平均荧光强度的判定阈值定为背景对照的两倍可以对这5种共8型病毒进行有效鉴定。通过多批次的实验,均能准确鉴定出上述5种病毒,没有出现交叉反应,且批次间的平均荧光值偏离不大,变异系数（CV）均小于8%,表明本方法的特异性和稳定性均较好。在空斑滴定病毒的基础上,对本方法的检测敏感性进行了验证,结果表明对乙脑和黄热病病毒的检测敏感性可达到1.4个PFU,对西尼罗病毒可达14个PFU。进而将本方法用于检测55份临床标本和传代标本,其检测结果和种特异RT-PCR方法相比,具有很高的一致性,而且其在混合样本的快速检测中具有显著的优势。结论通过设计合适的引物和探针,成功建立了可同时检测和分型5种虫媒病毒的悬浮芯片方法,为疾病的诊断和预防以及流行病学调查提供了新的手段。Objective To establish a multiplex suspension array for simultaneously detecting five species of important arbovirus： dengue virus （including four serotypes）, japanese B encephalitis virus, west nile virus, tick-borne encephalitis virus and yellow fever virus. Method All published genomic sequences of the above arboviruses were collected from GeneBank, and then, the genus universal primers and specific probes were designed according to the conservative and specific NS,5 region of the genus Flavivirus. The amine-modified probes were covalently coupled to different carboxylated mlcrospheres by carbodiimide methods. By combining general RT-PCR with virus-specific probe hybridization, a Luminex xMAP system-based suspension array, which could simultaneously differentiate five arboviruses and discriminate 1-4 serotypes of dengue virus, was successfully established. Results A cutoff value twice higher than that of the background fluorescence was judged as positive reaction. RT-PCR products of many arboviruses were used to evaluate the specificity and repeatability of the array, the results showed that the assay was highly specific and had good repeatability. Meanwhile, the coefficient of variability was less than 8% from inter- and intra- assay, implying that both the repeatability and stability of the array were good. Furthermore, several viruses which had been quantitated by plaque assay were used to determine the array＇s sensitivity. The results showed that the array＇s detection limits were about 1. 4 PFU for JBEV and YFV, 14 PFU for WNV respectively. 55 clinical and cultured samples were detected by the established assay, 16 out of the 55 samples were confirmed to be positive There was only one sample which was not detected contrasted with conventional RT-PCtL However, it had advantages in detecting complex samples rapidly. Conclusion A multiplex suspension array for simultaneously detecting and serotyping five species of important arboviruses has been successfully developed, and it may play an important r

关 键 词：虫媒病毒 悬浮芯片 检测 

分 类 号：R37-33[医药卫生—病原生物学]