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.
KEY WORDS: Nucleic acid-based amplification · NASBA · Diagnostics · IQ 2000 · Taura syndrome virus
Resale or republication not permitted without written consent of the publisherDis Aquat Org 73: [13][14][15][16][17][18][19][20][21][22] 2006 2002, Tang & Lightner 2005). In the CP1 and CP2 regions, low genetic variation (0 to 0.24% for CP1 and 0 to 0.35 or 0 to 5.6% for CP2 nucleotide sequences) was observed among the isolates analyzed. Due to the low-fidelity nature of the RNA-dependent RNA polymerase, new isolates of TSV that are able to replicate freely in a new species, such as the one described by Chang et al. (2004) in Penaeus monodon, are expected to keep emerging.Sensitive and specific detection of the causative agents is a prerequisite for effective disease prevention and management. Methods developed for TSV detection and characterization include histological analyses, immunoassay, in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR), and real-time RT-PCR (Lightner 1995, Mari et al. 1998, Nunan et al. 1998, Poulos et al. 1999, Castagna et al. 2004, Tang et al. 2004). Among the methods described above, diagnosis of potential TSV carriers or TSV outbreaks at early stages relies mainly on sensitive and specific platforms, such as the nested and real-time RT-PCR (Romano et al. 1995, Nunan et al. 1998, Tang et al. 2004). However, because of the extensive technical training and expensive equipment involved, these systems have not been widely adopted by farm operators for sensitive and timely on-site diagnosis of TSV.With the advantage of requiring only simple heating devices, various isothermal n...
