Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes.

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.

Specific detection of reverse transcription-loop-mediated isothermal amplification amplicons for Taura syndrome virus by colorimetric dot-blot hybridization.

The goal of this study was to develop a field diagnosis system based on isothermal reverse transcription-loop-mediated amplification (RT-LAMP) for shrimp Taura syndrome virus (TSV), placing emphasis on specific and simple detection of the LAMP amplicons. After a single-tube RT-LAMP reaction for TSV was established, colorimetric dot-blot hybridization (DBH) was adopted to detect signals only from the target-derived amplicons. The results showed that the modified DBH offered unambiguous and sensitive detection of the TSV RT-LAMP amplicons without the UV cross-linking and denaturation steps. Together, TSV RT-LAMP-DBH assay reached the same dilution point as reverse transcription-nested polymerase chain reaction-agarose gel electrophoresis (RT-nPCR-AGE) for TSV detection. Specificity of the assay was demonstrated by the absence of DBH signal from yeast tRNA and various shrimp viruses. TSV RT-LAMP-DBH was applied to 125 Penaeus vannamei and demonstrated a very good concordance (kappa value, 0.823) with RT-nPCR-AGE assay in detection efficiency. Furthermore, a one-step guanidinium thiocyanate (GuSCN) homogenization method was established to provide RNA extraction efficiency comparable to that of the TRIzol Reagent for RT-LAMP. Requiring simply a heating apparatus, the GuSCN RNA extraction-isothermal RT-LAMP-DBH protocol has the potential for further development for diagnosis of diseases in the field.

Rapid and sensitive detection of Taura syndrome virus using nucleic acid-based amplification.

Requiring only simple heating devices, isothermal nucleic acid-based amplification (NASBA) is a potential detection platform to be developed for on-site diagnosis of aquaculture pathogens. In this report, an NASBA assay has been developed for the Taura syndrome virus (TSV), one of the most devastating RNA virus pathogens for several penaeid shrimp species. The NASBA amplicons were detected by agarose gel electrophoresis and confirmed by Northern-blotting and dot-blotting analysis, using a biotinylated TSV-specific primer. The sensitivity of the TSV NASBA coupled with dot-blotting detection was approximately 5-fold less sensitive than that of the commercially available RT-nested, PCR-based IQ2000 TSV Detection and Prevention System that was also confirmed to be more sensitive than the RT-PCR-based TSV detection protocol recommended by the OIE (Office International des Epizooties). The specificity of the TSV NASBA reaction was substantiated by the results that RNA of non-target viruses did not generate any signals. Furthermore, a simple colorimetric microtiter plate assay employing TSV-specific capture and detection primers was developed as a simple alternative approach for the detection of NASBA amplicons. Taken together, the combination of the isothermal NASBA and colorimetric solid phase-based assays should allow sensitive, straightforward, and speedy on-site detection of TSV.