论文部分内容阅读
目的建立同时检测登革病毒(DENV1~4型)、日本脑炎病毒(JEV)、西尼罗病毒(WNV)、黄热病毒(YFV)、基孔肯雅病毒(CHIKV)、间日疟原虫(Pv)、恶性疟原虫(Pf)、三日疟原虫(Pm)和卵形疟原虫(Po)等6种12型病原体的悬液芯片检测方法。方法依据国家生物技术信息中心(NCBI)公开数据库中上述病原体的基因序列信息,设计并合成相关引物及探针,建立多重聚合酶链反应体系(多重PCR),产物与核酸探针微球组杂交后于Bio Plex200检测荧光信号值。结果建立的悬液芯片筛查方法具有较高的特异性和敏感性,能对6种12型病原体进行特异的检测。敏感性试验结果表明,Pm、CHIKV、Pv、WNV、DENV-1检测敏感性约为9DNA拷贝(3×10-7ng);Po、Pf、JEV、DENV-2、DENV-3、DENV-4和YFV检测敏感性约为90DNA拷贝(3×10-6 ng)。结论本研究建立的多重PCR技术结合悬液芯片筛查方法能快速、敏感、特异地同时检测6种共12型蚊媒传染病病原体,为疾病诊断及媒介生物携带病原体快速筛查和鉴定提供了新的手段。
Objective To establish a method for the simultaneous detection of Dengue virus (DENV1 ~ 4), Japanese encephalitis virus (JEV), West Nile virus (WNV), Yellow fever virus (YFV), Chikungunya virus (Pv), Plasmodium falciparum (Pf), Plasmodium malariae (Pm) and Plasmodium ovale (Po). Methods Based on the sequence information of the above pathogens in NCBI public database, primers and probes were designed and synthesized. Multiplex polymerase chain reaction (multiplex PCR) Fluorescence signal was detected on Bio Plex200. The results of the established suspension chip screening method has a high specificity and sensitivity, can be 6 kinds of 12-specific pathogen detection. Sensitivity test results showed that the detection sensitivity of Pm, CHIKV, Pv, WNV and DENV-1 was about 9 DNA copies (3 × 10-7ng); Po, Pf, JEV, DENV-2, DENV-3, DENV- YFV detection sensitivity of about 90 DNA copies (3 × 10-6 ng). Conclusion The multiplex PCR technique and suspension chip screening method established in this study can rapidly, sensitively and specifically detect six kinds of pathogens of total 12 types of mosquito-borne pathogens at the same time and provide the basis for disease diagnosis and rapid screening and identification of vector-borne pathogens New means.