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MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 112(7) July 2017
PAGES: 510-513 DOI: 10.1590/0074-02760160062 Technical notes
Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses

Felipe Gomes Naveca1+, Valdinete Alves do Nascimento1, Victor Costa de Souza1, Bruno Tardelli Diniz Nunes2, Daniela Sueli Guerreiro Rodrigues2, Pedro Fernando da Costa Vasconcelos2,3

1Fundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Manaus, AM, Brasil
2Ministério da Saúde, Secretaria de Vigilância em Saúde, Instituto Evandro Chagas, Ananindeua, PA, Brasil
3Unversidade do Estado do Pará, Belém, PA, Brasil

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Abstract

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.

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Financial support: FAPEAM PPP, DECIT/FAPEAM PPSUS, CNPq.
+ Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.
Received 21 February 2016
Accepted 29 June 2016