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Assessment of EvaGreen-based quantitative real-time PCR assay for enumeration of the microalgae Heterosigma and Chattonella (Raphidophyceae)

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Abstract

Heterosigma akashiwo and Chattonella species (Raphidophyceae) are difficult to detect and quantify in environmental samples because of their pleomorphic and fragile cell nature. In this study, we developed a quantitative real-time polymerase chain reaction (qRT-PCR) assay for the enumeration of these algal taxa using a new DNA-binding dye, EvaGreen. Species-specific qRT PCR primers to H. akashiwo, Chattonella antiqua, Chattonella marina, Chattonella ovata, and Chattonella subsalsa were designed to target the ITS2 rRNA gene intergenic region. Primer specificities were tested via BLAST searches. In addition, specificity was verified using empirical tests, including competitive PCR. The qRT PCR assay analyzing C t value and the log of cell number showed a significant linear relationship (r 2 ≥ 0.997). When light microscopy was used to monitor the population dynamics of targeted Raphidophyceae from Lake Shihwa, H. akashiwo was detected in ten samples and no Chattonella spp. were detected (70 samples collected from May, 2007 to January, 2008). In contrast, when the qRT-PCR assay was used, H. akashiwo was detected in 41 samples. C. antiqua, C. marina, and C. ovata were detected in eight samples. Most of the samples analyzed using qRT-PCR assays showed higher algal numbers than did those assayed via microscopy, suggesting that the enumeration of Raphidophyceae via classic microscopic methods most likely underestimates true algal concentration.

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Acknowledgements

We thank Prof. Yang-Ho Yoon for providing the Chattonella strain, You Kai for assistance with field work and Chloe Deodato for assistance with techniques. This study is supported by the National Research Foundation of Korea (NRF-2008-314-C00319) and Eco-STAR project program of the ministry of Korean Environmental Technology (EW21-07-12).

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  1. Department of Life Science and Research Institute for Natural Sciences, Hanyang University, Seoul, 133-791, South Korea

    Bum Soo Park & Myung-Soo Han

  2. Department of South Sea Research, KORDI, Geoje, 656-830, South Korea

    Seung Ho Baek

  3. Department of Green Life Science, College of Convergence, Sangmyung University, Seoul, 110-743, South Korea

    Jang-Seu Ki

  4. Department of Biology, University of Washington, Box 355325, Seattle, WA, 98195-5325, USA

    Rose Ann Cattolico

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Park, B.S., Baek, S.H., Ki, JS. et al. Assessment of EvaGreen-based quantitative real-time PCR assay for enumeration of the microalgae Heterosigma and Chattonella (Raphidophyceae). J Appl Phycol 24, 1555–1567 (2012). https://doi.org/10.1007/s10811-012-9816-2

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