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

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.

Fig. 1. : chimeric plasmid pOROV_MAYV. The pOROV_MAYV plasmid contains both Mayaro (MAYV) and Oropouche (OROV) targets in the context for in vitro RNA production from the T7 promoter site. The forward and reverse primer binding sites, as well as the probe binding sites are represented by a dark grey, light grey, and a black arrow, respectively. The pOROV_MAYV plasmid was ordered form IDT DNA Technology.

Fig. 2. : reverse transcription real-time polymerase chain reaction (RT-qPCR) with serial dilutions of the chimeric in vitro transcribed RNA containing both Mayaro (MAYV) and Oropouche (OROV) targets. Amplification plots for MAYV (A) and OROV (B), and linear regression for MAYV (C) and OROV (D) for ten-fold, 8-log, dilutions from 20 to 2E+08 copies, in duplicate. PCR efficiency in the multiplex assay was calculated with StepOnePlus Software v2.2 to be 98.642% [slope: -3.355, R2: 1] for MAYV, and 99.181% [slope: -3.341, R2: 1] for OROV. Ct = cycle threshold. Fluorescence values were exported to MS Excel and plotted using GraphPad Prism 6.0.