One-step reverse transcription-loop-mediated isothermal amplification assay for sensitive and rapid detection of porcine kobuvirus

Abstract

Porcine kobuvirus (PKoV) is associated with swine gastroenteritis, but its pathogenesis is uncertain. In this study, a rapid one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for the detection of PKoV is developed. A set of four primers specific to six regions within the PKoV 3D gene was designed for the RT-LAMP assay using total RNA extracted from PKoV-infected tissues. The reaction temperature and time for this assay were optimized. Compared with reverse-transcription PCR, RT-LAMP was able to detect PKoV at a 100-fold lower dilution. No cross-reaction was observed with other similar viruses, indicating that the assay is highly specific for PKoV. To investigate the prevalence of PKoV in symptomatic pigs in Sichuan province, the newly developed method was used to detect PKoV in a panel of clinical specimens, yielding a positive rate of 86.7% (144/166) in piglets. The results showed that the RT-LAMP assay is highly feasible in clinical settings. The data confirm that the RT-LAMP assay is rapid, simple and cost-effective and is particularly suitable for simple diagnosis of PKoV both in the field and in the laboratory.

Keywords: Detection; Porcine kobuvirus; Reverse transcription loop-mediated isothermal amplification (RT-LAMP).