DAO 99:215-225 (2012)  -  DOI: https://doi.org/10.3354/dao02476

Development of real-time PCR assays for discrimination and quantification of two Perkinsus spp. in the Manila clam Ruditapes philippinarum

Kousuke Umeda, Tomoyoshi Yoshinaga*

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
*‑Corresponding author. Email:

ABSTRACT: The Manila clam Ruditapes philippinarum is infected with 2 Perkinsus species, Perkinsus olseni and P. honshuensis, in Japan. The latter was described as a new species in Mie Prefecture, Japan, in 2006. Ray’s Fluid Thioglycollate Medium (RFTM) assay has been most commonly used to quantify Perkinsus infection. However, this assay cannot discriminate between species that resemble one another morphologically. We developed real-time PCR assays for the specific quantification of P. olseni and P. honshuensis. DNA was extracted using Chelex resin. Cultured P. olseni and P. honshuensis cells were counted and spiked into uninfected clam gill tissue prior to DNA extraction to generate standard curves, which allowed quantification based on the PCR cycle threshold values. We compared the RFTM assay with both real-time PCR assays by quantifying Perkinsus spp. in gill tissue samples from the same individual clams obtained from various localities in Japan. Infection intensities estimated by both assays were significantly correlated (r2 = 0.70). Our results suggest that the prevalence and infection intensity of P. honshuensis are much lower than for P. olseni in Manila clams.


KEY WORDS: Perkinsus olseni · Perkinsus honshuensis · qPCR · Ruditapes philippinarum · Manila clam · RFTM


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Cite this article as: Umeda K, Yoshinaga T (2012) Development of real-time PCR assays for discrimination and quantification of two Perkinsus spp. in the Manila clam Ruditapes philippinarum. Dis Aquat Org 99:215-225. https://doi.org/10.3354/dao02476

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