Adam J. Sepulveda
Renee M. Martin
Lacey R. Hopper
Patrick R. Hutchins
2018
<p><span>Conventional PCR is an established method to detect </span><i>Tetracapsuloides bryosalmonae</i><span>DNA in fish tissues and to confirm diagnosis of proliferative kidney disease (PKD) caused by </span><i>T. bryosalmonae</i><span>. 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 </span><i>T. bryosalmonae</i><span> 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 </span><i>T. bryosalmonae</i><span> infection status, PKX5f‐6r detected 35 positive samples, while the new assay detected 43. The discrepancy in </span><i>T. bryosalmonae</i><span> 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.</span></p>
application/pdf
10.1002/aah.10020
en
Wiley
Improved conventional PCR assay for detecting <i>Tetracapsuloides bryosalmonae</i> DNA in fish tissues
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