Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA)

• Published in: Diseases of aquatic organisms 2004-05, Vol.59 (2), p.93-100
• Main Authors: STARKEY, William G, MILLAR, Rose Mary, JENKINS, Mary E, IRELAND, Jacqueline H, MUIR, K. Fiona, RICHARDS, Randolph H
• Format: Article
• Language: English
• Subjects: Animal aquaculture; Animal productions; Animals; Biological and medical sciences; DNA Primers; DNA-Directed RNA Polymerases - metabolism; Fish Diseases - diagnosis; Fish Diseases - virology; Fishes; Fundamental and applied biological sciences. Psychology; Molecular Probe Techniques; Nodaviridae - genetics; Nodaviridae - isolation & purification; Nodavirus; Nucleic Acid Amplification Techniques - methods; Pisces; Pisciculture; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease H - metabolism; RNA Virus Infections - genetics; RNA Virus Infections - veterinary; RNA-Directed DNA Polymerase - metabolism; Sensitivity and Specificity; Vertebrate aquaculture
• ISSN: 0177-5103; 1616-1580
• DOI: 10.3354/dao059093

Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the co-ordinated activity of 3 enzymes: AMV reverse transcriptase, RNase H, and T7 RNA polymerase. We have developed a real-time NASBA procedure for detection of piscine nodaviruses, which have emerged as major pathogens of marine fish. Viral RNA was isolated by guanidine thiocyanate lysis followed by purification on silica particles. Primers were designed to target sequences in the nodavirus capsid protein gene, yielding an amplification product of 120 nucleotides. Amplification products were detected in real-time with a molecular beacon (FAM labelled/methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. Based on the detection of cell culture-derived nodavirus, and a synthetic RNA target, the real-time NASBA procedure was approximately 100-fold more sensitive than single-tube RT-PCR. When used to test a panel of 37 clinical samples (negative, n = 18; positive, n = 19), the real-time NASBA assay correctly identified all 18 negative and 19 positive samples. In comparison, the RT-PCR procedure identified all 18 negative samples, but only 16 of the positive samples. These results suggest that real-time NASBA may represent a sensitive and specific diagnostic procedure for piscine nodaviruses.