Development of loop-mediated isothermal amplification method for visualization detection of the highly virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV) in China

Abstract

A novel assay method to detect the highly virulent Porcine reproductive and respiratory syndrome virus (PRRSV) termed reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), was reported by using hydroxynaphthol blue (HNB) as the LAMP product colorimetric judgment. By the set of special primers, targeting the sequence that belongs to Nsp2 gene containing an 87 bp deletion mutation, the LAMP-based assay could be completed within 1 h at 63°C. Final concentration of 150 mM HNB was confirmed to be appropriate in LAMP product judgment without impact on the reaction system. The detection limit of the assay was 10³ copies per reaction, as determined by using a recombined plasmid. Genomes of various viruses were used to confirm the specificity of the LAMP-based assay, but only the highly virulent strains of PRRSV could be detected. All the internal organs from three stillborn piglets from a herd of pigs with PRRS-like symptoms were confirmed by LAMP-based assay to be infected not only with classical PRRSVs, but also the variant strains. These results suggest that the RT-LAMP assay with HNB provides a useful tool for the diagnosis of the highly virulent PRRSV infections in porcine rapidly.

Key words: Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), highly virulent porcine reproductive and respiratory syndrome virus (PRRSV), classical porcine reproductive and respiratory syndrome virus (PRRSV), hydroxynaphthol blue (HNB).