虫媒病毒性脑炎M-RT-PCR诊断方法的建立及应用

Design and application of M-RT-PCR diagnostic methods for arboviral encephalitis

目的 建立常见嗜神经性虫媒病毒 (arboviruses)分子生物学诊断方法及分子流行病学调查模型。方法 通过生物信息学方法筛选 3对常见嗜神经性虫媒病毒特异基因扩增引物 ,建立多重逆转录聚合酶链式反应 (multiplexreversetranscription PCR ,M RT PCR)体系。考察了体系的特异性、敏感性和广谱性并使用建立的M RT PCR体系对我国新分离的 3株病毒进行鉴定。结果 M RT PCR体系针对黄病毒及甲病毒敏感性达到 10 0PFU ,针对加利福尼亚脑炎病毒组达到 10 0 0PFU。利用M RT PCR体系证实 3株新分离嗜神经性虫媒病毒株中 2株为蜱传脑炎病毒远东型 ,另 1株为日本脑炎病毒Ⅰ型。结论 建立了针对嗜神经性虫媒病毒的分子生物学鉴定方法 ,该方法能够准确分析黄病毒属嗜神经性虫媒病毒 (日本脑炎病毒、蜱传脑炎病毒和西尼罗病毒 )的基因型、地理来源、进化及亲缘关系等 ,能够鉴定并区分甲病毒属不同病毒 (东方马脑炎病毒、西方马脑炎病毒和委内瑞拉马脑炎病毒 ) 。

Objective To establish a molecular biological diagnostic tool and a model of molecular epidemiological survey based on M-RT-PCR(multiplex revers transcription-PCR) for neuroinvasive arboviruses. Methods Three sets of primer pairs were selected for diagnosis of neuroinvasive arboviruses. A M-RT-PCR diagnostic method was established with those primers. After scrutinizing the specificity, sensitivity, and various strains were able to be identified by this method, and three new isolates were identified by M-RT-PCR. Results Visible product was observed for reactions containing 100PFU/ml, and no visible product for 10PFU/ml for SA-14, and 100PFU/ml for XJ-160, 1000 PFU/ml for LEIV-11554. Two new isolates from human brain tissue and ticks were considered as Far Eastern genotype of TBE. The other one was genotype Ⅰ of JEV. Conclusion Our M-RT-PCR diagnostic methods can be used for epidemiological analysis of flavivirus, identification of alphavirus and CEV.