一种无RNA纯化步骤TaqMan RT-PCR方法用于快速检测登革病毒

A TaqMan RT-PCR assay without RNA purification for rapid detection of dengue virus

目的 建立一种简便、快速的登革病毒核酸检测方法。方法 设计合成LNA探针，替代MGB探针，建立一步法TaqManRT—PCR方法，并用已知的登革病毒标准毒株进行方法的优化；用商品化的纯化试剂盒和简单的热释放法获取登革病毒细胞培养液和模拟含登革病毒血清中RNA。以优化的TaqManRT—PCR方法进行检测，比较有或无RNA纯化步骤检测的敏感性和重复性。结果 LNA探针TaqMan RT—PCR有较好的PCR扩增效率（104％）、较广的线性范围（10^0-10^-7样品稀释度）、较高的敏感性（0．003TCID50／反应）和较好的重复性（CV值小于4．53％），新方法检测经简单热释放RNA的细胞培养液和模拟含登革病毒血清中登革病毒也有较高的敏感性（0.03TCID50／反应）和较好的重复性（CV值小于6．36％）。结论 新型LNA探针可替代MGB探针用于TaqMan RT—PCR反应。简便的热释放RNA方法结合高敏感性的TaqMan RT—PCR可满足登革病毒快速检测的需要。

Objective To develop a simple and rapid detection method for nucleic acid of dengue virus. Methods A LNA probe was designed and synthesized to replace the MGB probe for the established TaqMan RT- PCR assay, and then optimized with a known dengue virus standard strain. A commercial RNA extraction kit and simple heat -release method were used to obtain dengue virus RNA from cell culture supernate and simulated dengue virus serum, which was detected by the optimized TaqMan RT - PCR assay. The results without RNA purification were compared with methods with RNA purification. Results The LNA probe based TaqMan RT- PCR assay showed good PCR amplification efficiency（ 104% ）, broad linear range（ 10^0 - 10 ^-7）, high sensitivity（0.003TCID50/reaction） and repeatability（ CV 〈 4.53% ）. High sensitivity （0.03TCID50/reaction） and repeatability（ CV 〈 6.36% ） were also observed by the new method for detection of the dengue virus in cell culture supernate and simulated serum after heat - releasing RNA. Conclusion The sensitive TaqMan RT - PCR assay, with innovated LNA probe to replace MGB probe, combined with simple heat - release RNA method, satisfied routine dengue virus detection.