title: Development and validation of an RNA- and DNA-based quantitative PCR assay for determination of Kudoa thyrsites infection levels in Atlantic salmon Salmo salar.
abstract: Quantitative PCR (QPCR) methods targeting the 18S rDNA gene (DNA QPCR) and cathepsin L mRNA (RNA QPCR) from Kudoa thyrsites (Gilchrist) were developed and compared with histology for determination of K. thyrsites infection levels in Atlantic salmon Salmo salar L. Both QPCR tests were specific, reproducible and sensitive down to 3 copies. DNA QPCR was able to detect lower K. thyrsites infection levels than those detected by RNA QPCR and histology. The higher sensitivity of the DNA-based test compared with the RNA-based test appeared to be biological in nature and suggested that when infection levels were low, there were fewer copies of cathepsin L mRNA than 18S rDNA genes. However, all 3 diagnostic methods were highly correlated. Regression analyses comparing DNA QPCR and histology data from 2 distinct groups of fish showed that the relationship between these 2 diagnostic methods was reproducible. A logistic regression analysis comparing diagnostic data with a visual assessment of post-mortem flesh quality indicated that histology was the single best predictor of flesh quality, followed by DNA QPCR and then RNA QPCR.
authors.affiliation: BC Centre for Aquatic Health Sciences, 621 Island Highway, PO Box 277, Campbell River, British Columbia V9W 5B1, Canada. val.funk@cahs-bc.ca
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Funk
authors.forename: Monique
authors.initials: M
authors.lastname: Raap
authors.forename: Ken
authors.initials: K
authors.lastname: Sojonky
authors.forename: Simon
authors.initials: S
authors.lastname: Jones
authors.forename: John
authors.initials: J
authors.lastname: Robinson
authors.forename: Christy
authors.initials: C
authors.lastname: Falkenberg
authors.forename: Kristina M
authors.initials: KM
authors.lastname: Miller
mesh_terms: D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000235:genetics; D004247:DNA / Q000032:analysis; D017931:DNA Primers / Q000737:chemistry; D056890:Eukaryota / Q000235:genetics / Q000302:isolation & purification*; D005393:Fish Diseases / Q000175:diagnosis* / Q000469:parasitology; D016015:Logistic Models; D008460:Meat / Q000592:standards; D009132:Muscles / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011529:Protozoan Infections, Animal / Q000175:diagnosis* / Q000469:parasitology; D012333:RNA, Messenger / Q000032:analysis; D012337:RNA, Ribosomal, 18S / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D020317:Salmo salar / Q000469:parasitology*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D004247:DNA; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L