7种非洲多发病毒病基于荧光微球的多元核酸检测方法的建立与初步评价  

A fluorescent bead-based multiplex PCR assay for the simultaneous detection of seven viral diseasesendemic in Africa

作　　者：闫芳域 殷强玲 李阿茜 芜为[1] 李川[1] 梁米芳[1] 李德新[1] 李建东[1] Yah Fangyu;Yin Qiangling;Li Aqian;Wu Wei;Li Chuan;Liang Mifang;Li Dexin;Li Jiandong(National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing , 100206, China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《中华实验和临床病毒学杂志》2018年第2期191-194,共4页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的建立非洲地区较为高发的7种重要病毒病基于荧光微球的多重核酸检测方法完成初步的评价分析。方法比较分析引起裂谷热、黄热，马尔堡，埃博拉、拉沙热、克里米亚-刚果出血热和基孔肯雅热等病原体的基因组序列，结合前期实时荧光PCR检测方法，设计合成特异性多重扩增引物和杂交探针，建立基于荧光微球的核酸检测方法，采用化学合成DNA和体外转录RNA等方法制备相关病原体模拟样本和参考品，评价检测方法的灵敏度和稳定性，利用30份登革热、汉城出血热等疑似患者标本和32份正常人血标本评价特异性。结果所建立的基于荧光微球的针对7种非洲多发病毒病核酸检测方法可特异性检出相应病原体核酸，检测限可达102～105拷贝/ μl，特异性为100%，组内变异系数均在12%以下，组间变异系数在15%以下，具有较高的稳定性。结论初步建立了基于荧光微球的7种非洲多发病毒病核酸检测方法，为相关疑似传染病筛查提供了新的选择。Objective To establish a fluorescent bead-based multiplex assay for the simultaneous detection of seven viral diseases endemic in Africa. Methods The genomie sequences of the viral pathogens causing Rift valley fever, Yellow fever, Marburg virus disease, Ebola virus disease, Lassa fever, Crimean- Congo hemorrhagic fever and Chikungunya fever were compared, PCR detection target fragments were selected, and amplification primers and hybrid probes were designed. The reference samples of related pathogens were prepared by chemical synthesis of DNA and in vitro transcription RNA. The sensitivity and stability of the detection method were evaluated. The specificity was evaluated by testing 30 samples of suspected dengue fever, and hantavirus diseases, and 32 healthy human blood samples. Results The fluorescent bead-based multiplex assay could specifically detect the corresponding pathogen, the detection limit was at a range of 102-105 copies/ μl1, the specificity was 100% , and the intra-assay coefficient of variation was below 12% , and the inter-assay coefficient of variation was below 15%. Conclusions A fluorescent bead-based multiplex PCR assay for the simultaneous detection of seven viral diseases endemic in Africa was established, which may provide a new choice for the screening of suspected infectious diseases.

关 键 词：病毒病 荧光微球 核酸检测 非洲 液相芯片 

分 类 号：R440[医药卫生—诊断学] R511[医药卫生—临床医学]