Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile

Background: Infectious Pancreatic Necrosis Virus (IPNV) is the etiological agent of a highly contagious disease that affects salmonids. In Chile, the second worldwide salmon producer, IPNV causes great economic loss and is one of the most frequently detected pathogens. Due to its high level of persistence and the lack of information about the efficiency of its diagnostic techniques, the National Reference Laboratory (NRL) for IPNV in Chile performed the first inter-laboratory ring trial, to evaluate the sensitivity, specificity and repeatability of the qRT-PCR detection methods used in the country. Results: Results showed 100% in sensitivity and specificity in most of the laboratories. Only three of the twelve participant laboratories presented problems in sensitivity and one in specificity. Problems in specificity (false positives) were most likely caused by cross contamination of the samples, while errors in sensitivity (false negatives) were due to detection problems of the least concentrated viral sample. Regarding repeatability, many of the laboratories presented great dispersion of the results (Ct values) for replicate samples over the three days of the trial. Moreover, large differences in the Ct values for each sample were detected among all the laboratories. Conclusions: Overall, the ring trial showed high values of sensitivity and specificity, with some problems of repeatability and inter-laboratory variability. This last issue needs to be addressed in order to allow harmonized diagnostic of IPNV within the country. We recommend the use of the NRL methods as validated and reliable qRT-PCR protocols for the detection of IPNV.

Differentiation of infectious pancreatic necrosis virus isolates by polymerase chain reaction.

Infectious pancreatic necrosis virus (IPNV) from lake trout of Cornwall lake (LT-IPNV), from trout of Jasper (Ja-IPNV) and Arctic char of Northwest Territories (AC-IPNV) are the three isolates recorded from western Canada. Two segments (nt 453-674 and nt 1197-1503) from VP2 coding region of RNA of the isolates were amplified by polymerase chain reaction (PCR) for differentiation. Primers for cDNA synthesis and amplification by PCR were chosen from VP2 coding region of RNA of Ja-IPNV. The segment of 453-674 could be amplified in all the three isolates at annealing temperature from 50 to 60 degrees C. However the segment nt 1197-1503 could be amplified only from Jasper and not from AC or LT-INV at primer annealing temperature ranging from 50 degrees-60 degrees C. PCR product could be obtained from the latter two isolates only at Primer annealing temperature of 45 degrees C. This difference in annealing temperature in PCR amplification between the isolates could be used for differentiating the isolates at genome level.