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Improved Conventional PCR Assay for Detecting Tetracapsuloides bryosalmonae DNA in Fish Tissues
Author(s) -
Hutchins Patrick R.,
Sepulveda Adam J.,
Martin Renee M.,
Hopper Lacey R.
Publication year - 2018
Publication title -
journal of aquatic animal health
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 52
eISSN - 1548-8667
pISSN - 0899-7659
DOI - 10.1002/aah.10020
Subject(s) - biology , amplicon , primer (cosmetics) , microbiology and biotechnology , dna , polymerase chain reaction , parasite hosting , gene , genetics , chemistry , organic chemistry , world wide web , computer science
Conventional PCR is an established method to detect Tetracapsuloides bryosalmonae DNA in fish tissues and to confirm diagnosis of proliferative kidney disease ( PKD ) caused by T. bryosalmonae . However, the commonly used PKX 5f‐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 , PKX 18s1266f‐1426r, is presented that demonstrates specificity, repeatability, and enhanced sensitivity over the PKX 5f‐6r assay. The limit of detection of the PKX 18s1266f‐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 PKX 5f‐6r. The PKX 18s1266f‐1426r also achieved 100% detection at sample DNA concentrations one order of magnitude lower than PKX 5f‐6r. Out of 127 salmonid fish with unknown T. bryosalmonae infection status, PKX 5f‐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.