[Show abstract][Hide abstract] ABSTRACT: Aiming to develop a rapid, low-cost, and user-friendly system for the diagnosis of white spot syndrome virus (WSSV), a PCR assay performed in capillary tubes under insulated isothermal conditions (iiPCR assay) was established on the basis of Rayleigh-Benard convection. WSSV amplicons were generated reproducibly within 30 min from a target sequence-containing plasmid in an iiPCR device, in which a special polycarbonate capillary tube (R-tube™) was heated isothermally by a copper ring attached to its bottom and shielded by a thermal baffle around its upper half. Furthermore, WSSV-specific amplicons were produced from nucleic acid extracts of WSSV-infected Penaeus vannamei in the WSSV iiPCR assay, with sensitivity comparable to that of an OIE-certified commercial nested PCR kit (IQ2000™ WSSV Detection and Prevention System). Specificity of the WSSV iiPCR assay was demonstrated as no amplicons were generated from shrimp genomic DNA, and IHHNV, MBV, and HPV DNA. iiPCR has a potential as a low-cost method for sensitive, specific and rapid detection of pathogens.
[Show abstract][Hide abstract] ABSTRACT: Aiming to establish a target amplicon-specific detection system for loop-mediated isothermal amplification (LAMP), the fluorescent resonance energy transfer (FRET) probe technology was applied to develop the FRET LAMP platform. This report describes the development of the first FRET LAMP assay targeting white spot syndrome virus (WSSV) of penaeid shrimp. A successful accelerated WSSV LAMP was assembled first in a conventional oven and confirmed by gel electrophoresis and dot-blot hybridization. Subsequently, two additional FRET probes designed to target one loop region within WSSV LAMP amplicons were added to the same LAMP reaction. The reactions were carried out in a LightCycler (Roche) and significant FRET signals were detected in real time. Optimization of the reaction using plasmid DNA shortened the time for the detection of 10(2) copies of the target DNA to less than 70min. Cross reactivity was absent with WSSV-free or infectious hypodermal and hematopoietic necrosis virus-infected Penaeus vannamei samples. The performance of this system was comparable with that of a nested PCR assay from 21 WSSV-infected shrimp. Specifically detecting target amplicons and requiring no post-amplification manipulation, the novel FRET LAMP assay should allow indisputable detection of pathogens with minimized risks of amplicon contamination.