A microfluidic-based system using reverse transcription polymerase chain reactions for rapid detection of aquaculture diseases

Abstract

This study presents an integrated microfluidic system capable of automatically performing four reverse-transcription polymerase chain reaction (RT-PCR) processes simultaneously for fast diagnosis of aquacultural diseases. This system integrates micro temperature control modules and a microfluidic control module. The micro temperature control modules have micro temperature sensors and array-type micro heaters that maintain precise and uniform temperature conditions for the RT-PCR processes. The microfluidic control module can automatically transport samples and reagents by using pneumatic micropumps, microvalves and microchannels. Moreover, by using random primers in the reverse-transcription (RT) process, the chip design is simplified and the consumption of RT products in the subsequent multiple polymerase chain reactions (PCR) is also minimized. After sample transport between the RT chamber and the PCR chambers is finished, the PCR process is then performed to amplify detection genes for each ribonucleic acid (RNA)-based virus. This microfluidic chip system, fabricated by using micro-electro-mechanical-system (MEMS) technology, has the following advantages, including high sensitivity, fast diagnosis, disposability, low reagent and sample consumption, portability, and low power consumption. Experimental data show that the developed system can successfully detect four types of purified RNA samples, including the nervous necrosis virus (NNV), Iridovirus, Vibrio anguillarum and the grouper Mx protein gene. The detection limit of the developed system is found to be 101 copies/μL, which is better than the limits of conventional methods which are approximately 102–103 copies/μL. The total reaction time for the detection of the four disease markers is experimentally found to be about 2.5 h. Consequently, the developed microfluidic system may provide a powerful tool for fast diagnosis of RNA-based viral vectors which cause aquaculture diseases.