qRT-PCR for enterovirus detection: Conversion to ultrafast protocols

Abstract Enterovirus group (EV) still causes significant morbidity with economic impact worldwide. Quantitative RT-PCR (qRT-PCR) technology offers many advantages over to conventional RT-PCR in terms of rapidity and specificity. The TaqMan hydrolysis probe technique and Syber Green I intercalating dye strategy are by then largely used. Several published protocols are applied routinely for EV detection in food and environmental analysis advanced in chemical strategies and thermal profiles, in order to reduce the response times. In this study an Ultra-Fast protocol to detect and quantify EV RNA genome was tested and the application prospective of the protocol was described and discussed. The assay effectiveness was evaluated comparing two different set of primers/probe, targeting a 5′UTR region of EV genome. Three different oligonucleotides concentration were tested: 200 nM, 250 nM and 300 nM for TaqMan technique whereas 200 nM, 300 nM and 400 nM were employed for Syber Green I chemistry. The results demonstrated the validity of this Ultra-Fast approach, compared to the Traditional and Fast protocols. The best performance was obtained using 200 nM of proper oligonucleotides, in both of the chemical strategies tested. The response time of analysis was reduced at 50′ (probe) and 57′ (intercalating dye) per run, against the other longer protocols. The oligonucleotides features can affect the assay performance and should satisfy specific characteristics.