基孔肯亚热、克里米亚-刚果出血热、裂谷热病毒核酸液相芯片检测方法的建立

Development of a liquichip method for detecting Chikungunya virus,Crimean Congo hemorrhagic fever virus and Rift Valley fever virus

目的建立基孔肯亚热、克里米亚-刚果出血热、裂谷热病毒核酸液相芯片检测方法。方法建立探针偶联至荧光微球、多重PCR扩增方法、杂交检测方法,并对所建立的液相芯片检测方法的灵敏度和特异性进行评价。结果建立的液相芯片检测方法能对基孔肯亚热、克里米亚-刚果出血热、裂谷热病毒中的任意1种、任意2种或者3种同时进行筛查检测,基孔肯亚热病毒检测的灵敏度为1×104copies/PCR,克里米亚-刚果出血热病毒检测的灵敏度为1×105copies/PCR,裂谷热病毒检测的灵敏度为1×103 copies/PCR。该方法对汉坦病毒、埃博拉病毒、黄热病毒、西尼罗病毒、马尔堡病毒的检测结果均为阴性。结论该方法具有高通量、多重检测、快速、敏感、特异的特点,为基孔肯亚热、克里米亚-刚果出血热、裂谷热病毒的筛查检测提供一种新方法。

Objective To establish a liquichip method which can detect Chikungunya virus, Crimean Congo hemorrhagic fever virus and Rift Valley fever virus simultaneously. Methods A liquichip method for simultaneously detecting of three species of arboviruses was developed and optimizated, including virus-specific nucleic probe coupling with microspheres of different codes, multiplex PCR, and hybridization. The sensitivity and specificity of the method were evaluated. Results The liquichip method can detect one, or two, or three species of the arboviruses in one reaction, with a high specificity and sentsitivity. The minimum detection limit for Chikungunya virus, Crimean Congo hemorrhagic fever virus and Rift Valley fever virus was l×10^4copies/PCR, 1×10^5 copies/PCR and 1 ×103^copies/PCR, respectively. Conclusion The method gives a high-throughput, multi-detection, rapid, accurate, sensitive detection of Chikungunya virus, Crimean Congo hemorrhagic fever virus and Rift Valley fever virus in the same tube, providing a new method for the screening and detecting of the three arboviruse.