An integrated microfluidic loop-mediated isothermal amplification platform for koi herpesvirus detection

An integrated microfluidic loop-mediated isothermal amplification (LAMP) platform consisting of a microfluidic chip and a portable operating system is proposed for koi herpesvirus (KHV) detection applications. The microfluidic chip is fabricated using a CO₂ laser system and comprises a sample chamber, two reagent chambers, a mixing chamber, a serpentine channel, a reaction chamber, and three flow rectifiers based on PDMS membranes. The main components of the operating system include a power source, a time relay, a microfluidic chip holder, four gas pressure valves, a microheater, and a temperature controller. In the proposed detection method, the KHV sample and LAMP reagent are loaded into the sample and reagent chambers, respectively, and are mixed under the effects of an external gas pressure driving force. The mixed solution is then heated at a temperature of approximately 63 °C for 60 min in order to replicate the KHV sample. Finally, the replicated sample is mixed with SYBR Green I dye in order to facilitate fluorescence-based KHV detection. The feasibility of the proposed microfluidic platform is confirmed by comparing the detection results obtained for a sample containing 10 ng/μL KHV with those obtained using a gel electrophoresis PCR-based method. The experimental results shows that the proposed LAMP platform has a KHV detection limit of around 3 × 10⁻² ng/μL. Overall, the results presented in this study show that the proposed microfluidic platform provides a compact, reliable and portable tool for KHV detection purposes.