A portable, 3D printed, microfluidic device for multiplexed, real time, molecular detection of the porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine deltacoronavirus at the point of need.

The porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) are emerging/reemerging coronaviruses (CoVs) of neonatal pigs that cause great economic losses to pig farms and pork processors. Specific, rapid, and simple multiplex detection of these viruses is critical to enable prompt implementation of appropriate control measures. Conventional methods for molecular diagnosis require skilled personnel and relatively sophisticated equipment, restricting their use in centralized laboratories. We developed a low-cost, rapid, semi-quantitative, field deployable, 3D-printed microfluidic device for auto-distribution of samples and self-sealing and real-time and reverse transcription-loop-mediated isothermal amplification (RT-LAMP), enabling the co-detection of PEDV, TGEV and PDCoV within 30 minutes. Our assay's analytical performance is comparable with a benchtop, real-time RT-LAMP assay and the gold standard quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay with limits of detection of 10 genomic copies per reaction for PEDV and PDCoV, and 100 genomic copies per reaction for TGEV. Evaluation of clinical specimens from diseased pigs with our microfluidic device revealed excellent concordance with both benchtop RT-LAMP and qRT-PCR. Our portable RT-LAMP microfluidic chip will potentially facilitate simple, specific, rapid multiplexed detection of harmful infections in minimally equipped veterinary diagnostic laboratories and on-site in pigs' farms.