Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses

Submitted by Raphael Rodrigues ([email protected]) on 2017-06-13T14:33:21Z No. of bitstreams: 1 ve_Felipe_Gomes_Naveca_etal_ILMD_2017.pdf: 611276 bytes, checksum: 7e501740aec08f9832c2d20d41b9f4a9 (MD5)Approved for entry into archive by Raphael Rodrigues ([email protected]) on 2017-06-13T14:42:56Z (GMT) No. of bitstreams: 1 ve_Felipe_Gomes_Naveca_etal_ILMD_2017.pdf: 611276 bytes, checksum: 7e501740aec08f9832c2d20d41b9f4a9 (MD5)Made available in DSpace on 2017-06-13T14:42:56Z (GMT). No. of bitstreams: 1 ve_Felipe_Gomes_Naveca_etal_ILMD_2017.pdf: 611276 bytes, checksum: 7e501740aec08f9832c2d20d41b9f4a9 (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus, AM, Brasil.Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus, AM, Brasil.Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus, AM, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Unversidade do Estado do Pará. Belém, PA, Brasil.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