Development of a Handheld Microfluidic Chip for On-Site Multiplex Detection of Four Porcine Diarrhea-Related Virus

The porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), and porcine circovirus type 2 (PCV2) of neonatal pigs cause great economic losses to pig farms and pork processing enterprises. 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. A low-cost, rapid, and field-deployable handheld chip was developed for visual codetection of PEDV, TGEV, PoRV, and PCV2 within 60 min based on colorimetric loop-mediated isothermal amplification (LAMP). The assay's analytical performance is comparable with that of a real-time RT-LAMP assay and the gold standard quantitative reverse transcription polymerase chain reaction (RT-PCR) assay with limits of detection of 100 genomic copies for all PEDV, TGEV, PoRV, and PCV2. Evaluation of clinical specimens from diseased pigs using our handheld chip revealed excellent concordance with both benchtop LAMP and RT-PCR. The portable handheld LAMP microfluidic chip will potentially facilitate simple, specific, and rapid multiplexed detection of infection disease in minimally equipped veterinary diagnostic laboratories and on-site in pigs' farms.