Infectious salmon anaemia virus (ISAV) ringtest: ...
|Title||Infectious salmon anaemia virus (ISAV) ringtest: validation of the ISAV diagnositic process using virus-spiked fish tissues and ISAV TaqMan® real-time RT-PCR|
|Author(s)||F. S. B. Kibenge, M. J. T. Kibenge, E. Masaoud|
|Journal||Journal of Aquaculture Research and Development|
|Abstract||Fourteen laboratories validated their procedure for detection of infectious salmon anaemia virus (ISAV) in fish tissues using TaqMan® real-time RT-PCR targeting ISAV RNA segment 8. The participants included 12 laboratories from South America, one from Asia and one from Europe. The OIE Reference Laboratory for ISA at the Atlantic Veterinary College in Canada served as the standard. All laboratories received a panel of 36 blind-coded samples representing six different ISAV preparations in homogenized fish liver or L-15 medium, each in six replicates. A clearly positive control ISAV of known titer was also included. From the results obtained, the 14 laboratories that submitted results reported no false positives. False negatives were mostly observed in the ISAV-spiked fish liver homogenate samples. The lowest virus titer to be detected in the fish liver homogenate was 101 TCID50/ml, but the virus titer that could be detected accurately by most laboratories was 103 TCID50/ml in L-15 medium. Within those laboratories that accurately detected presence of virus in a sample, there was great variation in the Ct values making it impossible to recommend a single cut-off Ct value. A significant factor influencing the Ct values obtained and therefore the diagnostic sensitivity might be the thermocycler software used. The repeatability of the test within each laboratory was high, but the reproducibility between laboratories was low. Presumably this could be improved if all the laboratories used the same RNA extraction method since the starting quality and the quantity of the RNA template is the main determinant of the quality of results once reagents have been optimized. The low reproducibility of the test between laboratories is also suggestive of the need to standardize the threshold fluorescence line of the thermocycler software and to use properly trained personnel to perform the test.|
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