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Moving molecular diagnostics from laboratory to clinical application: a case study using infectious pancreatic necrosis virus serotype A
Author(s) -
Kerr C.R.C.,
Cunningham C.O.
Publication year - 2006
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2006.01885.x
Subject(s) - infectious pancreatic necrosis virus , serotype , virology , titer , biology , virus , reverse transcription polymerase chain reaction , real time polymerase chain reaction , gene , messenger rna , biochemistry
Aims: Using an RT‐PCR method for detection of infectious pancreatic necrosis virus (IPNV) in Atlantic salmon as a model, this study examined the optimization and validation required to provide a method suitable for IPNV detection from fish tissue. Methods and Results: IPNV‐positive Atlantic salmon kidney samples that had been titred or kidney spiked with IPNV were used. The amount of RNA in the reverse transcription (RT), RT denaturation temperature and incubation time, PCR annealing temperature and number of cycles were optimized. The optimized RT‐PCR was able to detect IPNV in Atlantic salmon kidney calculated to have a titre of ten infectious units. Conclusions: Extensive optimization is required to produce a PCR for detection of fish pathogens from methods designed in the laboratory. Significance and Impact of the Study: This study demonstrated some of the many variables that should be optimized before a fully validated assay can be claimed and illustrates the extensive validation required to fulfil requirements of the OIE and is of relevance to laboratories carrying out clinical testing.