Portable DNA extraction and loop-mediated isothermal amplification (LAMP) for the on-site detection of white spot syndrome virus (WSSV)

Abstract

White spot disease (WSD) has been the most severe and lethal threat to the global shrimp industry for the past three decades. WSD caused by white spot syndrome virus (WSSV) can result in severe economic losses in shrimp culture. The disease can cause rapid and mass mortality of 90–100 % within 3–10 days. Currently, there are no effective treatment measures available for the control of WSD. Strict biosecurity, possible elimination, and early diagnosis can prevent the spread of the pathogen. Several advanced diagnostic methods have been standardised for the early and rapid detection of WSD. However, the field deployable diagnostic methods are limited. LAMP is a cost-effective, alternative nucleic acid amplification method that operates under isothermal conditions. In this study, a novel field-portable LAMP was developed for the diagnosis of WSSV. The WSSV amplification was optimized at an isothermal condition of 65 °C and a reaction time of 55 min. In this closed-tube amplification, the end results were easily visualized with the naked eye by observing the simple color change. This assay is highly sensitive and can detect up to 6 copies of WSSV plasmid DNA and 0.1 fg of WSSV genomic DNA. Additionally, this diagnostic method is highly specific and did not cross-react with other shrimp pathogens such as IHHNV, IMNV, EHP and Vibrio parahaemolyticus or host DNA of Penaeus vannamei, P. monodon, and P. indicus. Furthermore, a field-deployable DNA (FDD) extraction method has been standardised with a simple rotor and dry bath. This visual LAMP-coupled FDD extraction did not require any sophisticated equipment or technical expertise. Thus this robust LAMP assay can potentially be used for routine surveillance, point-of-care testing, early detection, and timely intervention of WSD.