Improved Conventional PCR Assay for Detecting Tetracapsuloides bryosalmonae DNA in Fish Tissues.

Abstract

Conventional PCR is an established method to detect Tetracapsuloides bryosalmonaeDNA in fish tissues and to confirm diagnosis of proliferative kidney disease (PKD) caused by T. bryosalmonae. However, the commonly used PKX5f-6r primers were designed with the intention of obtaining sequence information and are suboptimal for determining parasite DNA presence. A new PCR assay to detect T. bryosalmonae 18s rDNA, PKX18s1266f-1426r, is presented that demonstrates specificity, repeatability, and enhanced sensitivity over the PKX5f-6r assay. The limit of detection of the PKX18s1266f-1426r assay at 95% confidence was 100 template copies, and the new primers detected parasite DNA more consistently at template concentrations below 100 copies than did PKX5f-6r. The PKX18s1266f-1426r also achieved 100% detection at sample DNA concentrations one order of magnitude lower than PKX5f-6r. Out of 127 salmonid fish with unknown T. bryosalmonae infection status, PKX5f-6r detected 35 positive samples, while the new assay detected 43. The discrepancy in T. bryosalmonae detection between the two primer sets may be attributed to several differences between the assays, including oligonucleotide melting temperatures, the use of a touchdown PCR thermal cycle, and amplicon length.