Rapid Amplification and Detection of Foodborne Pathogenic Rotavirus by Continuous-flow Reverse Transcription-Polymerase Chain Reaction Integrated with Online Fluorescence Analysis

Abstract

Abstract A continuous-flow polymerase chain reaction (PCR) microfluidic system integrated with reverse transcription (RT) of RNA and online fluorescence analysis was fabricated in this work and successfully applied for the fast amplification and identification of foodborne pathogenic rotaviruses. In this continuous-flow RT-PCR device, isothermal copper blocks provided the required temperatures for RT-PCR, and a polytetrafluoroethylene capillary was used as the RT-PCR reaction channel. By using circulating water to cool the annealing zone, annealing temperatures as low as 31 °C could be obtained for the herein-presented device, and good temperature control and stability was also achieved. Therefore, this device could be used for different PCR protocols. Online fluorescence analysis of the PCR products, which was integrated into this device, was subsequently carried out. When the flow rate was 7.5 mm s −1 , amplification of the cDNA reverse transcribed from rotavirus RNA and the subsequent online fluorescence analysis of the amplification products could be completed in about 12 min (about 10 min for amplification and about 2 min for analysis). By using this microfluidic device, the RT-PCR of rotavirus RNA and the online fluorescence analysis of its products could be accomplished within approximately 1 hour, with an RNA detection limit of 6.4 × 10 4 copies μL −1 . This study provides a new biochemical method for the rapid diagnosis of RNA-based viruses.