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.
Teng
Ping-Hua
PH
Farming IntelliGene Technology Corporation, 1-2, Gung-Ye Seventh Road, Tai-Chung Industrial Park, Tai-Chung 407, Taiwan.
Chen
Chu-Liang
CL
Wu
Cheng-Nan
CN
Wu
Sih-Ying
SY
Ou
Bo-Rong
BR
Lee
Pei-Yu
PY
D000818:Animals; D017756:Aquaculture / Q000379:methods*; D015152:Blotting, Northern / Q000662:veterinary; D003124:Colorimetry / Q000662:veterinary; D015342:DNA Probes / Q000737:chemistry; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D015151:Immunoblotting / Q000662:veterinary; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary*; D033561:Penaeidae / Q000821:virology*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D013624:Taiwan; D013997:Time Factors
D015342:DNA Probes