分型悬液芯片检测疟原虫的方法建立及应用  

Development and application of liquid bead array method for the detection of plasmodium

作　　者：史玲莉[1] 闫冀焕[1] 李云[1] 罗明[2] 沈军[1] 韩伟[3] 

机构地区：[1]河北国际旅行卫生保健中心,河北石家庄050000 [2]北京市疾病预防控制中心 [3]河北出入境检验检疫局

出　　处：《中国国境卫生检疫杂志》2015年第2期90-94,共5页Chinese Journal of Frontier Health and Quarantine

基　　金：国家质检总局科研基金项目(2012IK225)

摘　　要：目的建立一种基于悬液芯片技术的疟原虫检测和分型方法,对4种疟原虫进行快速检测和鉴定。方法依据国家生物技术信息中心(NCBI)公开数据库上4种疟原虫的特异管家基因序列信息,设计并合成相关引物探针序列。抽提核酸DNA,经PCR扩增,产物与核酸探针微球组杂交后于Bio Plex200检测荧光信号值。结果优化了PCR方法,间日疟原虫(Pv)、三日疟原虫(Pm)的悬液芯片检测敏感性约9DNA拷贝,恶性疟原虫(Pf)、卵形疟原虫(Po)的悬液芯片检测敏感性约90DNA拷贝。进而将本方法用于检测30份临床样本,其检测结果与分型特异荧光PCR方法完全一致,而且在混合感染样本的快速检测中具有显著的优势。结论建立的可同时检测4种疟原虫的悬液芯片方法特异性高,省时,无交叉反应。为快速筛查和鉴定疟原虫提供了新的手段。Objective To establish a method for detecting plasmodium by liquid bead array,and detect and identify four kinds of plasmodium with fast speed. Methods Primers and probes were designed and synthesized according to genomic sequences in the NCBI Genbank database. Protozoa DNA was extracted and target sequences were amplified. PCR products were hybridized with coupled nucleic acid probe beads set and detected by the Bio-PlexTM200 system. Results PCR conditions were optimized,and the limits of detection for each plasmodium were about9 DNA copies for Pv、Pm and 90 DNA copies for Pf、Po.30 clinical samples were detected by the established assay,12 out of the 30 samples were confirmed to be positive. Compared with the specific fluorescence real-time PCR, it had the very high consistency. However,it had obvious advantages in detecting complex samples rapidly. Conclusion A liquid bead array method for detecting plasmodium was developed,which was a highly specific and saving time way,and had no cross-reaction to simultaneously detect four kinds of plasmodiu. It plays a new and important role in rapid screening and identification of plasmodium.

关 键 词：疟原虫 悬液芯片 PCR 

分 类 号：R115[医药卫生—公共卫生与预防医学]