Cross-platform evaluation of commercial real-time reverse transcription PCR master mix kits using a quantitative 5′nuclease assay for Ebola virus

Selection of optimal reaction master mix reagents is essential to obtain the best performance with diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) assays Every year the number of commercially available master mix kits increases so it is prudent to periodically evaluate kits on the market In this study we evaluated five commercial real-time RT-PCR master mix kits the RealMasterMix RT-PCR ROX kit the AgPath-ID One-Step RT-PCR kit the SuperScript III Platinum One-step Quantitative RT-PCR system the QuantiTect Probe RT-PCR lot and the LightCycler RNA HybProbe amplification kit using a 5'nuclease assay for Ebola virus The kits were evaluated using the manufacturer s recommended conditions as well as conditions which have been used with the Ebola virus assay during outbreaks When evaluated for use in Ebola virus RNA detection the AgPath-ID kit resulted in the greatest sensitivity in comparison to the other four lots The efficacy of the AgPath-ID kit was instrument-independent in the five real-time PCR platforms tested This study demonstrated that Ebola virus RNA detection was not equivalent among the master mix reagents studied and thus that this variable can affect real-time RT-PCR assay sensitivity Furthermore this study rates the master mix reagents for their suitability providing diagnostic laboratories the option to select from these kits to suit their specific laboratory needs for real-time RT-PCR Published by Elsevier Ltd