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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 112, No. 7, 2017, pp. 510-513
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Bioline Code: oc17068
Full paper language: English
Document type: Note
Document available free of charge
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Memórias do Instituto Oswaldo Cruz, Vol. 112, No. 7, 2017, pp. 510-513
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Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
Naveca, Felipe Gomes; Nascimento, Valdinete Alves do; Souza, Victor Costa de; Nunes, Bruno Tardelli Diniz; Rodrigues, Daniela Sueli Guerreiro & Vasconcelos, Pedro Fernando da Costa
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.
Keywords
real-time PCR; Mayaro virus; Oropouche virus; Amazon
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