A Microfluidic Diagnostic Device Capable of Autonomous Sample Mixing and Dispensing for the Simultaneous Genetic Detection of Multiple Plant Viruses

摘要

As an efficient approach to risk management in agriculture, the elimination of losses due to plant diseases and insect pests is one of the most important and urgent technological challenges for improving the crop yield. Therefore, we have developed a polydimethylsiloxane (PDMS)-based microfluidic device for the multiplex genetic diagnosis of plant diseases and pests. It offers unique features, such as rapid detection, portability, simplicity, and the low-cost genetic diagnosis of a wide variety of plant viruses. In this study, to realize such a diagnostic device, we developed a method for the autonomous dispensing of fluid into a microchamber array, which was integrated with a set of three passive stop valves with different burst pressures (referred to as phaseguides) to facilitate precise fluid handling. Additionally, we estimated the mixing efficiencies of several types of passive mixers (referred to as chaotic mixers), which were integrated into a microchannel, through experimental and computational analyses. We first demonstrated the ability of the fabricated diagnostic devices to detect DNA-based plant viruses from an infected tomato crop based on the loop-mediated isothermal amplification (LAMP) method. Moreover, we demonstrated the simultaneous detection of RNA-based plant viruses, which can infect cucurbits, by using the reverse transcription LAMP (RT-LAMP) method. The multiplex RT-LAMP assays revealed that multiple RNA viruses extracted from diseased cucumber leaves were successfully detected within 60 min, without any cross-contamination between reaction microchambers, on our diagnostic device.