Naveca Felipe Gomes, Nascimento Valdinete Alves do, Souza Victor Costa de, Nunes Bruno Tardelli Diniz, Rodrigues Daniela Sueli Guerreiro, Vasconcelos Pedro Fernando da Costa
Fundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Manaus, AM, Brasil.
Ministério da Saúde, Secretaria de Vigilância em Saúde, Instituto Evandro Chagas, Ananindeua, PA, Brasil.
Mem Inst Oswaldo Cruz. 2017 Jul;112(7):510-513. doi: 10.1590/0074-02760160062.
We describe a sensitive method for simultaneous detection of Oropouche and Oropouche-like viruses carrying the Oropouche S segment, as well as the Mayaro virus, using a multiplexed one-step reverse transcription real-time polymerase chain reaction (RT-qPCR). A chimeric plasmid containing both Mayaro and Oropouche targets was designed and evaluated for the in vitro production of transcribed RNA, which could be easily used as a non-infectious external control. To track false-negative results due to PCR inhibition or equipment malfunction, the MS2 bacteriophage was also included in the multiplex assay as an internal positive control. The specificity of the multiplex assay was evaluated by Primer-Blast analysis against the entire GenBank database, and further against a panel of 17 RNA arboviruses. The results indicated an accurate and highly sensitive assay with amplification efficiency greater than 98% for both targets, and a limit of detection between two and 20 copies per reaction. We believe that the assay described here will provide a tool for Mayaro and Oropouche virus detection, especially in areas where differential diagnosis of Dengue, Zika and Chikungunya viruses should be performed.
我们描述了一种灵敏的方法，该方法使用多重一步逆转录实时聚合酶链反应（RT-qPCR）同时检测携带奥罗普切S片段的奥罗普切病毒和类奥罗普切病毒，以及马亚罗病毒。设计并评估了一种包含马亚罗和奥罗普切靶标的嵌合质粒用于体外转录RNA的生产，其可轻松用作非感染性外部对照。为追踪因PCR抑制或设备故障导致的假阴性结果，多重检测中还加入了MS2噬菌体作为内部阳性对照。通过针对整个GenBank数据库的Primer-Blast分析以及进一步针对一组17种RNA虫媒病毒评估了多重检测的特异性。结果表明该检测准确且高度灵敏，两个靶标的扩增效率均大于98%，每个反应的检测限在2至20个拷贝之间。我们认为本文所述的检测方法将为马亚罗病毒和奥罗普切病毒的检测提供一种工具，尤其是在需要对登革热、寨卡病毒和基孔肯雅病毒进行鉴别诊断的地区。
