LAMP Assay Coupled with a Pyrococcus furiosus Argonaute System for the Rapid Detection of Porcine Epidemic Diarrhea Virus.

Abstract

Porcine epidemic diarrhea virus (PEDV) infection can lead to serious acute intestinal infectious disease, bringing huge economic losses to the pig industry. In addition to triggering an extremely high mortality rate for lactating piglets, there is currently a lack of effective treatments and vaccines. Therefore, rapid, accurate, sensitive, and specific detection of PEDV is critical for timely control. In this study, a nucleic acid detection method combining reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Pyrococcus furiosus Argonaute (PfAgo) was established for the detection of PEDV and performed after optimizing the system (mainly for the design and screening of the LAMP primers and PfAgo gDNA). The optimized system had a detection limit as low as 2.4 copies/μL. To reach more timely on-site detection of PEDV and overcome the reliance on bulky and complex equipment, a lateral flow strip was introduced into the system, which could detect the target as low as 24 copies/μL. This RT-LAMP-PfAgo system took about 35 min to react, and the results could be observed and clarified with the naked eyes. Moreover, the method was highly specific and had no cross-reactivity with other swine pathogens. The detection results for the clinical samples were consistent with those obtained by the gold standard method, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), proving its applicability. In conclusion, the established RT-LAMP-PfAgo system can provide a new solution for the development of a portable, visual PEDV testing platform.