2013 Volume 48 Issue 2 Pages 42-47
PCR assays were developed for the rapid and sensitive detection of Azumiobodo hoyamushi, the protistan pathogen that causes soft tunic syndrome in the cultured ascidian Halocynthia roretzi. Two sets of A. hoyamushi-specific primers based on the 18S rRNA and β-tubulin gene sequences, respectively, of the flagellate were designed and validated for the detection of A. hoyamushi. In both PCR assays, the flagellate genes were detected in diseased ascidians, but not in apparently healthy ones. When DNA were extracted from flagellate suspensions obtained from infected tunics, the detection limits of the PCR assays using primers for the 18S rRNA and β-tubulin were 3.4 × 10 cells/mL and 3.4 × 102 cells/mL, respectively. Even at the early stage of infection when the slight clinical signs were observed only in the siphons, A. hoyamushi was detected from the affected tissue using 18S rRNA PCR, but not β-tubulin PCR. Thus, the 18S rRNA PCR is a rapid, sensitive and specific assay for diagnosing soft tunic syndrome.
