Comparison and evaluation of Renibacterium salmoninarum quantitative PCR diagnostic assays using field samples of Chinook and coho salmon

Abstract

Renibacterium salmoninarum is a Gram-positive bacterium causing bacterial kidney disease (BKD) in susceptible salmonid fishes. Several quantitative PCR (qPCR) assays to measure R. salmoninarum infection intensity have been reported, but comparison and evaluation of these assays has been limited. Here, we compared 3 qPCR primer/probe sets for detection of R. salmoninarum in field samples of naturally exposed Chinook and coho salmon first identified as positive by nested PCR (nPCR). Additional samples from a hatchery population of Chinook salmon with BKD were included to serve as strong positive controls. The 3 qPCR assays targeted either the multiple copy major soluble antigen (msa) genes or the single copy abc gene. The msa/non-fluorescent quencher (NFQ) assay amplified R. salmoninarum DNA in 53.2% of the nPCR positive samples, whereas the abc/NFQ assay amplified 21.8% of the samples and the abc/TAMRA assay 18.2%. The enzyme-linked im-munosorbent assay (ELISA) successfully quantified only 16.4% of the nPCR positive samples. Although the msa/NFQ assay amplified a greater proportion of nPCR positive samples, the abc/NFQ assay better amplified those samples with medium and high ELISA values. A comparison of the geometric mean quantity ratios highlighted limitations of the assays, and the abc/NFQ assay strongly amplified some samples that were negative in other tests, in contrast to its performance among the sample group as a whole. These data demonstrate that both the msa/NFQ and abc/NFQ qPCR assays are specific and effective at higher infection levels and outperform the ELISA. However, most pathogen studies will continue to require multiple assays to both detect and quantify R. salmoninarum infection.

Bench‐top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

“…Within the operating range of the qPCRs, there was less than a 1 log 10 difference at a given seeding level in quantity estimates between the qPCRs that detect sequences of the msa gene (qPCR #1 and #2) and the qPCR that detects a sequence of the single‐copy abc transporter permease gene (PCR #3), despite the use of R. salmoninarum isolates with differing msa gene copy numbers in the tests. In contrast to the conclusion of Sandell & Jacobson (), our results suggested that in practical application any of the three qPCRs could be used for estimating R. salmoninarum quantities and that factors such as extraction and PCR efficiencies, pipetting errors, or starting R. salmoninarum DNA copy numbers may have more effect on quantification accuracy.…”

“…; Faisal & Eissa ; Nance et al . ; Sandell & Jacobson ). A variety of factors have been hypothesized to impact assay concordance.…”

“…; Faisal & Eissa ; Nance et al . ; Sandell & Jacobson ). Experimental exposure of a group of fish to R. salmoninarum on a single date as occurred in our study may result in a reduction or skewing of the spectrum of infection stages manifested in the population.…”

“…; Nance et al . ; Sandell & Jacobson ). Correlation between qPCR and ELISA values is usually stronger at higher infection levels, but exceptions have been noted (Sandell & Jacobson ).…”

“…; Halaihel et al . ; Sandell & Jacobson ), none has been sufficiently extensive or rigorous to fully validate a particular method according to standardized published procedures (Westgard ; OIE ). Comparisons frequently have been made using tissues from fish of unknown R. salmoninarum infection status, making it difficult to evaluate non‐concordant results among assays to determine which results actually represent ‘true positives’ and ‘true negatives.’…”