拉克罗斯病毒和雪靴野兔病毒双重RT-PCR方法的建立

目的建立针对拉克罗斯病毒(LAC)和雪靴野兔病毒(SSH)的双重RT-PCR检测方法。方法分别针对LAC和SSH病毒的S基因设计2对特异性引物,建立同时检测LAC和SSH的双重RT-PCR方法。以黄病毒属的黄热病毒(YFV)、库京病毒(KUNV)、登革病毒(DENV)、乙型脑炎病毒(JEV),布尼亚病毒属的塔希纳病毒(TAHV)和甲病毒属的巴马哈森林病毒(BFV)、玛雅罗病毒(MAYV)、盖塔病毒(GETV)为模板验证方法的特异性,以不同拷贝数的病毒RNA检测该方法的敏感性。结果建立的双重RT-PCR方法能同时扩增到LAC和SSH的目的片段;敏感度分别达到LAC1.41×105copies/μl,SSH 5.6×104copies/μl;与YFV、KUNV、BFV、MAYV无交叉反应。结论建立了双重RT-PCR方法,可用于LAC和SSH病毒的快速检测。

Development of duplex RT-PCR for La Crosse virus and Snowshoe hare virus

Objective To develop a duplex RT-PCR method for detecting of La Crosse virus （LAC） and snowshoe hare virus （SSH） simultaneously. Methods Two sets of specific primers were designed for S gene of LAC and SSH, which were used in one PCR reaction. The PCR conditions were optimized. The specificity was verified by using 8 kinds of viruses, including Kunjin Virus（KUNV）, Yellow fever virus（YFV）, Dengue virus（DENV）, Japanese encephalitis virus （JEV）, Tohyna virus（TAHV）, Barmah Forest virus（BFV）, Mayaro virus（MAYV） and Getah virus（GETV）. The sensitivity was detected by using different copy of viral RN＆ Results The specific PCR products of LAC and SSH were amplified. The sensitivity was 1.41×10^5 copieshxl and 5.6×10^4copies/μl, respectively. None of other viruses was found positive by duplex RT-PCR. Conclusion The duplex RT-PCR method had been developed, through which LAC and SSH could be detected.