Quantification of Microcystis in a Eutrophic Lake by Simple DNA Extraction and SYBR Green Real-time PCR

• Published in: Microbes and Environments Vol. 23; no. 4; pp. 306 - 312
• Main Authors: Tomioka, Noriko, Nagai, Takashi, Kawasaki, Tatsuya, Imai, Akio, Matsushige, Kazuo, Kohata, Kunio
• Format: Journal Article
• Language: English
• Published: Japan Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions / Japanese Society for Extremophiles 01.01.2008; Japan Science and Technology Agency
• Subjects: DNA extraction; eutrophic lake; Freshwater; Microcystis; quantification; SYBR Green I; Japan, Honshu, Ibaraki Prefect., Kasumigaura L
• ISSN: 1342-6311; 1347-4405
• DOI: 10.1264/jsme2.ME08515

A simple extraction method and real-time PCR with SYBR-Green I were combined to monitor Microcystis 16S rRNA gene (16S rDNA) concentrations over a wide range. A Fast DNA SPIN Kit (MP Biomedicals) was used to extract rDNA quantitatively. Real-time PCR amplified Microcystis rDNA for quantification with the forward primer Micro229f, which was newly designed by us and was highly specific for Microcystis, and the reverse universal primer 342r. The method developed here can detect Microcystis at concentrations as low as 3 cells mL-1. The rDNA concentration and cell count were highly correlated in the range from 1.2×104 to 1.1×106 copies mL-1 and from 9.5×102 to 1.0×105 cells mL-1, respectively, in a canal where Microcystis algal blooms occur annually. The Microcystis rDNA concentration in Lake Kasumigaura was measured by the application of our method to water samples collected monthly from April 2004 to March 2006. Microcystis was not detected by microscopy from January to June 2005, except in May, but our method detected 1.0×103 to 1.0×104 copies mL-1 of Microcystis rDNA during this period. This result clearly showed that our method is useful for clarifying the annual fluctuation in Microcystis concentration, especially when concentrations are low.