Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification.
We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and 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. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100x more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.
Starkey
William G
WG
Department of Virology, Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK. w.g.starkey@stir.ac.uk
Smail
David A
DA
Bleie
Hogne
H
Muir
K Fiona
KF
Ireland
Jacqueline H
JH
Richards
Randolph H
RH
D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D005393:Fish Diseases / Q000175:diagnosis / Q000821:virology*; D046829:Isavirus / Q000235:genetics / Q000302:isolation & purification*; D007668:Kidney / Q000821:virology; D009976:Orthomyxoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012367:RNA, Viral / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D020317:Salmo salar / Q000821:virology*; D021161:Self-Sustained Sequence Replication / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013997:Time Factors
D017931:DNA Primers; D012367:RNA, Viral