Integrated microfluidic reverse transcription-polymerase chain reaction for rapid detection of food- or waterborne pathogenic rotavirus

Abstract

The development of microfluidic tools for nucleic acid analysis has become a burgeoning area of research during the postgenome era. Here we have developed a microfluidic device that integrates reverse transcription (RT) and polymerase chain reaction (PCR) with online fluorescence detection to realize a rapid detection system for performing both genetic amplification and product analysis. The microfluidic device mainly comprises a grooved copper heating block for RT and a heated cylinder for amplification. To expedite the analysis process, we combined the continuous-flow PCR with an online fluorescence detection system that allows analysis of amplification products within 1 min. Rotaviruses are worldwide enteric pathogens in humans and animals responsible for a significant burden of disease through person-to-person transmission and exposure to contaminated foods and water. In this study, rotavirus from stool specimens was successfully amplified and detected using the RT-PCR microfluidic system within 1 h, and the limit of detection of the RNA concentration was estimated to be 3.6 × 10 4 copies μl −1. Compared with a large-scale apparatus, the integrated microfluidic system presented here can perform rapid nucleic acid amplification and analysis, possibly making it a crucial platform for future diagnosis application.