Instrument-free, visual and direct detection of porcine reproductive and respiratory syndrome viruses in resource-limited settings.

Abstract

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most complicated and dangerous diseases in pigs with high mortality since it modulates the immune system of the lungs and has been closely associated with secondary infection of other lethal bacteria and viruses. The gold standard of molecular diagnosis for PRRSV, reverse transcription (RT)-PCR, is time-consuming, expensive and requires transportation of samples to a specialized laboratory. In this study, a direct colorimetric RT-loop-mediated isothermal amplification (RT-LAMP) method was developed to specifically and rapidly detect PRRSV. The RT-LAMP outcomes can be visualized by the naked eye after 45 min of incubation at 65˚C without any cross-reactivity recorded with the bacteria and other viruses tested. In particular, the mobile, non-instrumented, commercial pocket hand warmers were demonstrated to su-ccessfully provide constant temperature for consistent nucleic acid amplification throughout the RT-LAMP reactions. The limit of detection of the assay was defined as the genomic RNA concentration extracted from a known viral titer of 10-2.5 TCID50/ml. The direct use of clinical serum samples required a simple dilution to maintain the performance of the colorimetric RT-LAMP assay. Therefore, the direct colorimetric RT-LAMP assay developed is well-qualified for producing a ready-to-use kit for PRRSV diagnosis in the field. Keywords: porcine reproductive and respiratory syndrome; rapid testing; RT-LAMP; colorimetric; direct detection; instrument-free.