Evaluation of Molecular Biomarkers in Quantitative Determination of the Toxic Population of Microcystis in Environmental Samples
Microcystis spp. are known to have toxic and nontoxic cells even in an environmental sample. Cell counts of Microcystis can not give a correct measure of toxin risk. From the toxic strains of Microcystis, series of DNA sequences (mcyA~mcyJ) have been identified as genes of microcystin synthetase, known to be responsible for the biosynthesis of microcystins, a group of cyclic heptapeptidic hepatotoxins. In order to distinguish quantitatively the toxic and nontoxic populations of Microcystis in environmental samples, we have tried to identify the specific DNA segments of toxic strains and to develop a real time quantitative PCR for the unambiguous measurement of toxinogenic Microcystis in a low cyanobacteria biomass of water bodies. Six characteristic segments representing the genes of mcyA, mcyB, mcyC, mcyD, mcyE and the promoter within this microcystin synthetase gene cluster were selected for this Q-PCR amplification using specially-designed primers. The method was first evaluated using 8 toxic and 4 non-toxic clones of M. aeruginosa isolated from aquaculture ponds and water reservoirs in Taiwan and then applied in the environmental monitoring. Facts obtained are, (i) the selected amplicons were present in all toxic clones but absent or less amount in non-toxic clones; (Ⅱ) segment of mcyD from the toxic clones showed consistent Tm values, implying this mcyD segment the most conserved one among the tested mcys; (iii) a linear correlation existed between the microscopically determined cell numbers and the PCR threshold cycles; (iv) gene copy within a cell showed variable and even reach a number of 20 at different growth phase in an enriched medium; (v) gene copy within a cell remained as one in the stationary phase of a culture or in environmental samples. Our data suggested that the selection of suitable primer pairs for specific mcy segment is important to avoid the misleading.
Jian-Zhi Lin Hong-Nong Chou
Institute of Fisheries Science, National Taiwan University, Taipei 10617, Taiwan
国际会议
武汉
英文
13
2011-04-05（万方平台首次上网日期，不代表论文的发表时间）