iso-μmGene: Isothermal amplification-based portable microfluidic system for simple, reliable and flexibly multiplexed Genetic identification and quantification
Simple, reliable and flexibly multiplexed genetic identification and quantification of microbial pathogens is in urgent need for early disease diagnosis and timely treatment. This study presented an isothermal amplification-based portable microfluidic system (iso-μmGene) with features of multi-well chips for convenient filling and reliable sealing, flexible detection throughput, and stand-alone and well-performing point of care (POC) genetic testing device. Using disposable chips with two kinds of reaction wells (eighteen and ten wells) and a device prototype with independent four chip holders, the iso-μmGene enables on-demand analysis of different target genes in one sample per chip and one to four samples (chips) per run, requiring only a single pipetting step for dispensing per chip with dehydrated primers. To completely seal the Loop-Mediated Isothermal Amplification (LAMP) reaction system to minimize the risk of amplicon escape, a dedicated plastic shell is used to assemble the array-type chip and reliably close its openings. Meanwhile, to enhance the precision for flexibly multiplexed detection and decrease the size and cost of the device, we designed a thermoelectric cooler (TEC)-based temperature-control module including two separate units and a CCD-based fluorescence imaging module containing a linear translation stage for real-time LAMP assay. This work demonstrated applications for parallel detection of 2～2000 CFU (Colony-Forming Units) per reaction well with good intra- and inter-chip reproducibility using the crude lysates of two aquacultural pathogens Edwardsiella tarda and Vibrio harveyi. Overall, the iso-μmGene presented here possesses both sophisticated instrument’s functionality and performance and POC device’s portability and cost.