Foundation And Application Of PCR-based Detection Of Microcystin Synthetase Gene

Microcystis can frequently form as noxious blooms in freshwater, are of major concern because many strains produce cyclic heptapeptide toxins called microcystins. Microcystins are a kind of endotoxins, which are strong carcinogens. Drinking water-contained microcystins for a long time may cause liver damages on human beings or other animals.Common detection measures, such as HPLC and ELISA, can detect microcystins in water. But they cannot monitor the producing of microcystins before they are released from the cell of Microcystis. Specific primers of microcystin synthetase gene B (mcyB) were designed and selected in this study. The intact cell PCR-mediated detection was established. This detection would not be affected by different living stages of algae and interference of other algae and bacteria. In 50 L reaction system with 10 uL sample, the sensitivity of one step PCR was 100-1000 cells/reaction, the sensitivity of nested PCR was 1-10 cells/reaction. This result showed that we had established a sensitive and effective detection on mcyB.Thirty Microcystis strains were isolated from 11 reservoirs in Guangdong province, and were detected with above method. These reservoirs were Hedi reservoir, Tangxi reservoir, Liuxihe reservoir, Dajingshan reservoir, Shatian reservoir, Gaozhou reservoir, Qiyeshi reservoir, Feilaixia reservoir, Shenzhen reservoir, Xinfengjiang reservoir and Chishijing reservoir. These 30 strains include three species, which are Microcystis aeruginosa, Microcystis marginata and Microcystis jlos-aquae. Twelve positive strains were detected in all 30 strains and the positive ratio was 40%. The resources of positive strains were Hedi reservoir, Tangxi reservoir, Liuxihe reservoir, Dajingshan reservoir, Gaozhou reservoir, Feilaixia reservoir and Shenzhen reservoir.PCR-mediated detection of mcyB gene was used to detect water samples from 12 supply water reservoirs. These 12 reservoirs included 11 reservoirs mentioned above and Dashahe reservoir. There were 136 samples collected from July 2003 to March 2004. Fifteen positive samples were detected by one step PCR and the total positive ratio was 11.1%. Fifty-nine positive samples were detected by nested PCR and the total positive ratio was 43.4%.There were positive samples in all reservoirs except Shenzhen reservoir. There were high positive ratios by nested PCR of water samples from Tangxi reservoir, Gaozhou reservoir, Dajingshan reservoir and Shatian reservoir. The positive ratios of these four reservoirs were 95.2%, 66.7%, 66.7% and 60% respectively. It suggested that Microcystis with potential microcystin-producing ability widely existed in these four reservoirs. Therefore these four reservoirs should be mainly detected. Thepositive ratio of one step PCR in Tangxi reservoir had been 47.6%. This result implied that the concentration of Microcystis, which has potential microcystin-producing ability in positive samples, was high. It had been over 1 X 105 cells/L. More attention must be paid to this reservoir.