双重荧光RT-PCR检测4种致病性亚型埃博拉病毒方法的建立

目的建立可同时检测苏丹、扎伊尔、本迪布焦和科特迪瓦4种致病性亚型埃博拉病毒的双重荧光RT-PCR检测方法。方法根据4种致病性亚型埃博拉病毒的核蛋白NP基因保守序列,针对苏丹型/扎伊尔型病毒以及针对本迪布焦型/科特迪瓦型病毒,相应设计2套引物和探针。以体外转录的4种亚型埃博拉病毒RNA为模板,进行条件的优化以及方法特异性、灵敏度、重复性试验,建立双重荧光RT-PCR检测方法。结果新建立的双重荧光RT-PCR方法检测只对4种埃博拉病毒阳性对照RNA模板有特异性扩增,与肾综合征出血热病毒、登革病毒、黄热病毒、西尼罗病毒、日本脑炎病毒、基孔肯雅病毒均无交叉反应,2套引物和探针的检测灵敏度均可达到最低50拷贝/μL。苏丹型和本迪布焦型埃博拉病毒阳性对照RNA模板的4种稀释度（2×108、2×106、2×104、2×102拷贝/μL）重复检测3次均有较好的重复性。结论建立的方法能同时检测上述4种亚型埃博拉病毒,灵敏度高,特异性强,可用于埃博拉病毒疑似病例的检测。

Development of a duplex fluorescence RT-PCR assay for detecting four pathogenic subtypes of Ebola virus

Objective To develop a duplex fluorescence RT-PCR assay for detecting four patho- genic subtypes of Ebola virus, including the subtypes of Sudan, Zaire, Bendi bujiao and Ivory Coast. Methods Based on the conserved sequence of nucleoprotein （NP） gene of four subtypes of Ebola virus, two sets of primers and probes were designed corresponding to the Sudan/Zaire subtype and to Bendi bu- jiao/Ivory Coast subtype. Using the Ebola virus RNA of four subtypes transcribed in vitro as templates, the reaction condition were optimized and the specificity, sensitivity, and reproducibility were tested. Then a duplex fluorescence RT-PCR assay was developed. Results The specificity of the method was high on de- tection the Ebola virus nucleic acid. There were no cross reactions with hemorrhagic fever with renal syn- drome virus, Dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus and Chikungu- nya virus. Detection sensitivities of two sets of primers and probes can reach a minimum of 50 copies/μL. Duplicate detections for three times had better repeatability at four dilutions （ 2×10^8, 2 ×10^6 , 2 ×10^4, 2 ×10^2 copies/μL） of positive control RNA templates of subtypes of Sudan and Bendi bujiao. Conclusion The duplex fluorescence RT-PCR assay can simultaneously detect the four subtypes of Ebola virus with high sensitivity and specificity, and can be used for detection on suspected cases of Ebola virus infection.