Detection Of Microcystis Based On PC-IGS Sequence

The increasing incidence of mass developments of cyanobacteria in fresh- and brackish water in whole-world is a matter of growing concern due to the production of toxins that threaten human and livestock health. Microcystis is the main producer of toxin-producing cyanobacteria. Technology for detecting cyanobacteria commonly use microscope to identify morphological characteristics, which requires the operator is very familiar with the cell morphology of various cyanobacteria, otherwise, it is easy to miss or false detect. Therefore, the accuracy of conventional microscopy is not high, and developing cyanobacteria, especially Microcystis early monitoring, analysis and early warning system is of great significance.The development of modern biotechnology and vast amounts of biological information in genetic database can offer the possibility of establishing rapid and effective methods to detect a target genotype. PCR method of detecting genes has been reported whereas it is easy to produce false. With respect to nucleic acid molecular hybridization's obvious advantages of high sensibility and specificity, the conjugation of DNA probes with hybridization can be used for environmental monitoring, having advantages in high-speed and sensitivity.Therefore,the goal of the research is to establish a molecular biological method for early warning and forecast of Microcystis, which is based on PC-IGS sequence of Microcystis. In this thesis, the main contents and conclusions are as follows.1. The available PC-IGS (phycocyanin intergenic spacer) sequences are used for base comparison analysis and building phylogenetic tree. The Microcystis and Anabena strains form a monophyletic cluster respectively with respect to PC-IGS sequence. It is testified that PC-IGS sequence is conservative highly in cyanobacteria, and can used to establish molecular biological methods of detecting cyanobacteria. Pairs of high specific probes (capture probe and signal probe) are designed based on the PC-IGS sequence for the division Microcystis.2. The ingredients and concentration of hybridization solution, washing buffer, temperature, time of hybridization and so on hybridization parameters are optimized through many times tests, and the ELSHA method for monitoring Microcystis is established successfully. The correctness of the ELSHA was proved by the results of hybridization for Microcystis and Anabaena cultured in the laboratory. Then absorbance at 405nm of ELSHA is plotted against the cell concentration in the hybridization system to establish the calibration curve (y=0.00002x+0.1225,R2=0.9883) for ELSHA,which achieves quality detection for water samples. The minimal detectable concentration by ELSHA is only 1cells/mL.3. It is consistent that results of detecting the Microcystis concentration in water samples from GuanQiao Lake, Jing Lake, Yuan Lake, YuJia Lake by ELSHA, microscope and PCR. Further more, the relative standard deviation (RSD) by microscope examination maybe 20.0%, which is higher than 14.1% by ELSHA. Therefore, ELSHA developed in the study is a rapid and effective method for Microcystis bloom early warning and forecast.