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

AUTOR(ES)

Naveca, Felipe Gomes, Nascimento, Valdinete Alves do, Souza, Victor Costa de, Nunes, Bruno Tardelli Diniz, Rodrigues, Daniela Sueli Guerreiro, Vasconcelos, Pedro Fernando da Costa

FONTE

Mem. Inst. Oswaldo Cruz

DATA DE PUBLICAÇÃO

2017-07

RESUMO

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.

Documentos Relacionados

• A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus
• Detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection
• Multiplexed, Real-Time PCR for Quantitative Detection of Human Adenovirus
• Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics
• Expression Profiling of the Whole Arabidopsis Shaggy-Like Kinase Multigene Family by Real-Time Reverse Transcriptase-Polymerase Chain Reaction1