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 reversetranscription 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

Fast identification of foodborne pathogenic viruses using continuous-flow reverse transcription-PCR with fluorescence detection

“…The developed microfluidic system not only overcomes the thermal cross-talk effects of radiation and/or conduction but also utilizes fluorescence microscopy to realize the on-line product detection. Up to now, the amplification products of most microfluidic RT-PCR (Felbel et al 2008;Hartung et al 2009;Lee et al 2008;Liao et al 2005;Lien et al 2007;Lien et al 2009;Tsai and Sue 2006) have been analyzed by agarose gel electrophoresis and ethidium bromide staining, which is time-consuming and labor-intensive. Therefore, our current efforts are focused on the on-line fluorescence detection, which offers several obvious advantages.…”

“…Quake and co-workers (Marcus et al 2006b) were one of the first groups to demonstrate microchip RT-PCR in a single chamber. Although there have been a few demonstrations Hsieh et al 2009;Huang et al 2006;Kaigala et al 2008;Lee et al 2008;Lien et al 2007;Lien et al 2009;Marcus et al 2006a, b;VanDijken et al 2007) of chamber stationary RT-PCR microfluidics, they generally lack the flexibility to change the reaction rate, resulting in more cycling and heating time. Moreover, in order to reduce the reaction time and power consumption, the system thermal mass often needs to be optimized considerably (Liu et al 2002).…”

Micro real-time PCR device using a circulation pump

“…However, standard PCR techniques using the end-point detection method has drawbacks such as poor precision, difficult quantitation, and low sensitivity, among others (Richard et al 2003). As such, the standard PCR device is rapidly being replaced by miniaturized realtime PCR devices in order to meet the requirements of fast analysis, portability, and low cost (Xiang et al 2005;Neuzil et al 2006;Lien et al 2009;Hettiarachchi et al 2012).…”

A disposable, integrated loop-mediated isothermal amplification cassette with thermally actuated valves

“…Although valveless technologies have been developed to reduce evaporation (Zhang and Xing 2010), valves are still predominantly used for sealing in microfluidics (Chen et al 2007;Wang et al 2006;Das et al 2007;Oh and Ahn 2006). Diverse, ingenious actuation mechanisms for microfluidic valves have been developed such as piezoelectric (Li et al 2004;Shao et al 2004), magnetic (Bae et al 2002;Gaspar et al 2008), thermal (Kim et al 2004;Chen et al 2008), pneumatic (Lagally et al 2004;Lien et al 2009), and hydraulic (Oh et al 2005). Thermal actuation, which does not require moving components, is often adopted in microfluidic chips because of its simplicity, low cost, and reliability (Liu et al 2004(Liu et al , 2002Chen et al 2005;Wang et al 2005).…”