Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with colorimetric gold nanoparticle (AuNP) probe assay for visual detection of Penaeus vannamei nodavirus (PvNV)

Abstract

Penaeus vannamei nodavirus (PvNV) is an emerging viral infection that has caused muscle necrosis or a white tail disease in cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei, leading to loss in the productions. Rapid detection of PvNV is essential for further control disease. A combination between reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay and colorimetric gold nanoparticle (AuNP) probe was developed for rapid, sensitive and inexpensive detection of PvNV in this study. RT-LAMP reaction successfully detected PvNV at 63°C for 45 and 5 min for hybridization of LAMP amplified product, followed by salt-induced AuNP-labelled ssDNA probe aggregation to visual colour development. This method showed identical results to LAMP followed by gel electrophoresis and spectrophotometric detection, and it was 10 times sensitive more than conventional nested RT-PCR. The new method revealed negative results with other shrimp pathogens. This study provides the direct visualization of LAMP reaction by AuNP probe hybridization, that significantly reduces the time and cost required for the molecular diagnostic of infectious diseases and offers to use in aquaculture health management.

Significance and impact of the study: This study represents the first report of Penaeus vannamei nodavirus (PvNV) detection using the colorimetric RT-LAMP technique. The application of RT-LAMP assay combined with colorimetric gold nanoparticle (AuNP) probe to detect the PvNV offers simple, rapid and sensitive technique and does not require sophisticated equipment with applicable for small or field laboratories. This method can further employ to screen broodstock before breeding, to screen postlarvae before ponds stocking, to monitor shrimp in rearing ponds and to assess the occurrence of this virus in shrimp farming.