摘要
建立多重检测东方马脑炎病毒、西方马脑炎病毒、委内瑞拉马脑炎病毒、基孔肯亚病毒、登革病毒、日本脑炎病毒、黄热病毒、西尼罗病毒、圣路易斯脑炎病毒、版纳病毒、布尼亚病毒、裂谷热病毒的悬浮芯片方法。将十二种蚊媒病毒分为3组，每组使用标记有不同anti—TAG的上游引物及标记有生物素的下游引物对分别构建多重PCR体系，PCR产物与偶联了x—TAG的编码磁性微球进行杂交，最后利用Bio—plex100分析系统对12种蚊媒病毒分别进行单一、多重液相芯片检测。将平均荧光强度的判定阈值定为背景对照的3倍，多批次实验均能对十二种单一病毒样本和混合病毒样本进行准确鉴定，未出现交叉反应，表明该方法特异性较好。敏感性测试结果显示本检测方法具有高灵敏度。通过利用偶联有TAG及生物素的引物对，成功建立了可同时检测12种蚊媒病毒的液相芯片检测技术平台。该平台对于病原体的检测具有较好的应用前景。
This paper describes the development of a multiplexed x-TAG suspension arrays for simultaneous detection of mosquito-borne virus including Eastern equine encephalitis virus （ EEEV ）, Western equine encephalitis virus （ WEEV ）, Venezuelan Equine Encephalitis Virus （VEEV）, Chikungunya virus （CHIK）, Dengue virus （DEN） , Japanese Encephalitis Virus （ JEV ）, Yellow fever virus （ YF）, West Nile virus （ WN ）, St. louis encephalitis virus （ SLEV ）, Banna virus （ BAV ）, Bunyavirus （BUN） , and Rift Valley fever virus （RVF）. The 12 species viruses were divided into three groups, and multi- PCR primers were designed by groups with up-primers labeled by anti-TAG on 5＇ end and down-primers labeled by biotin on 5＇ end separately. The PCR amplicons were then hybridized with the magnetic microspheres coupled with x-TAG, and results analysed on Bio-plex 100. Reactions with the mean fluorescence intensity （MFI） three times higher than that of the background was judged as positive reactions. The results of single plex and multiplex assays for the virus cultures demonstrated specific positive signals without cross reaction, and the sensitivity and specificity of the method were satsifying. The liquid biochip technology platform with the beads coupled with TAG was established which can simultaneously detect 12 types of mosquito-borne viruses, indicating an application potential for pathogen multiplex detection.
出处
《寄生虫与医学昆虫学报》
CAS
2013年第4期244-250,共7页
Acta Parasitologica et Medica Entomologica Sinica
基金
国家科技部国际合作项目（2010DFA34250）
国家“十二五”科技支撑计划项目（2013BAD12B03）
关键词
悬浮芯片
蚊媒病毒
多重检测
X-TAG
Suspension array
Mosquito-borne virus
Simultaneous detection
x-TAG
