title: Comparison of quantitative PCR and ELISA for detection and quantification of Flavobacterium psychrophilum in salmonid broodstock.
abstract: A quantitative PCR (qPCR) assay was developed for Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease. The assay was targeted to fp1493 as it encodes a putative outer membrane protein (FP1493) that is reactive to the monoclonal antibody (MAb FL43) used in a standardized F. psychrophilum capture enzyme-linked immunosorbent assay (ELISA). The qPCR was specific to F. psychrophilum and was able to detect between 8 and 809000 copies of fp1493. To determine if antigen level in the tissue was indicative of bacterial concentration, kidney samples from 108 steelhead Oncorhynchus mykiss and coho salmon O. kisutch female broodstock were screened by ELISA and qPCR. There was no correlation between ELISA optical density (OD) values and the number of F. psychrophilum cells g⁻¹ of kidney tissue as estimated by qPCR (rS = 0.42; p > 0.05). The median number of F. psychrophilum cells in steelhead samples was 6.11 × 10³ cells g⁻¹ of tissue. For coho salmon samples, the median number of cells was 3.95 × 10³ cells g⁻¹ of tissue. Agreement between the 2 assays was less than 50%. As fp1493 is a single-copy gene and differential expression of FP1493 has been reported, we hypothesize that the discrepancy between the 2 assays is due to increased expression of FP1493 in the in vivo environment. Therefore, ELISA OD values most likely provide an indication of differential protein expression, while the qPCR assay estimates bacterial load in tissue.
authors.affiliation: Department of Fish & Wildlife Sciences and the Aquaculture Research Institute, University of Idaho, PO Box 441136, Moscow, ID 83844-1136, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Long
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Call
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Cain
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005260:Female; D005393:Fish Diseases / Q000175:diagnosis / Q000382:microbiology*; D045826:Flavobacteriaceae Infections / Q000175:diagnosis / Q000662:veterinary*; D005417:Flavobacterium / Q000302:isolation & purification*; D017686:Oncorhynchus mykiss; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity