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°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.
KEY WORDS: Nodavirus · NASBA · RT-PCR · Diagnostics · Nucleic acid amplification
Resale or republication not permitted without written consent of the publisherDis Aquat Org 59: [93][94][95][96][97][98][99][100] 2004 ular diagnostic methods based on RT-PCR (Nishizawa et al. 1994, Grotmol et al. 2000 or nested RT-PCR (Dalla Valle et al. 2000) have been developed for betanodaviruses, and have contributed significantly to the diagnosis and control of VNN. However, these assays are relatively time-consuming, may be compromised by limited sensitivity, and are susceptible to false positive reactions arising from amplicon contamination.Nucleic acid sequence based amplification (NASBA) (Compton 1991) is an isothermal method for nucleic acid amplification that is particularly suited to RNA targets (Kievits et al. 1991). Diagnostic procedures based on NASBA methodology have been described for several viruses including Human Immunodeficiency Virus Type 1 (de Baar et al. 1999), cytomegalovirus (Witt et al. 2000, enterovirus (Heim & Schumann 2002), West-Nile and St Louis Encephalitis viruses (Lanciotti & Kerst 2001), parainfluenza virus (Hibbitts et al. 2003) and hepatitis C virus (Damen et al. 1999). In the NASBA procedure, target-specific amplification is achieved through oligonucleotide primers and the co-ordinated activity of 3 enzymes: reverse transcriptase, RNase H, and T7 RNA polymerase. The final amplification product is a singlestranded RNA, the polarity of which is opposite to that of the target. Real-time detection in NASBA can be performed using...
