Journal: Memórias do Instituto Oswaldo Cruz
Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses
Figure Lengend Snippet: : 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.
Article Snippet: We used the TaqMan® Fast Virus 1-Step master mix (Applied Biosystems) for RT-qPCR amplification with the recommended cycling parameters, 50ºC for 5 min for reverse transcription, 95ºC for 20 s for RT inactivation/initial denaturation, followed by 45 cycles of 95ºC for 3 s and 60ºC for 30 s. RNA (2 μL) was used as a template in a 20 µL reaction, and all assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems).
Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, In Vitro, Amplification, Polymerase Chain Reaction, Multiplex Assay, Software, Fluorescence, Mass Spectrometry