水中有毒蓝藻的荧光定量PCR检测法

发布时间：2018-05-11 17:03

本文选题：荧光定量PCR + 有毒蓝藻　；参考：《环境与健康杂志》2016年02期

【摘要】：目的建立水中有毒蓝藻的荧光定量PCR检测方法。方法取20 ml纯培养藻液和500 ml采集水样用0.22μm滤膜进行过滤,收集藻体提取DNA,应用SYBR Green对微囊藻毒素合成酶基因mcy A进行实时荧光定量PCR检测,根据软件获取的以模板标准品与Ct值建立的标准曲线,计算样品中的mcy A基因拷贝数和产微囊藻毒素(MC)的蓝藻的浓度。结果在13.8~1.38×106拷贝/L的线性范围内,所得回归方程为y=-3.45 lgx+36.2,r=0.989 9。方法检出限为10.7拷贝/L,加标回收率为88.4%~106.0%,RSD为3.6%~4.0%。应用该方法和传统的显微镜检法对实验室纯培养产毒铜绿微囊藻的检测结果呈正相关(r=0.994 0,P0.05)。结论该方法灵敏度高、精确度高、检测范围宽、重复性好,适合用于蓝藻水华暴发的早期预警。
[Abstract]:Objective to establish a fluorescence quantitative PCR method for the detection of toxic cyanobacteria in water. Methods 20 ml pure culture algae solution and 500ml water sample were filtrated with 0.22 渭 m filter membrane, and the microcystin-synthase gene mcy A was detected by real-time fluorescence quantitative PCR (PCR), and the microcystin synthase gene mcy A was detected by SYBR Green. According to the standard curve obtained by software, the copy number of mcy A gene and the concentration of cyanobacteria producing microcystins were calculated. Results in the linear range of 13.8 ~ 1.38 脳 10 ~ (6) copies / L, the regression equation was obtained as follows: yaw-3.45 lgx ~ (36.2) / L ~ (0.989 9). The detection limit was 10.7 copies 路L ~ (-1) and the recovery rate was 88.4% and 106.0% RSD was 3.6% and 4.0% respectively. There was a positive correlation between this method and the traditional microscope method for the detection of microcystis aeruginosa produced by pure culture in laboratory. Conclusion this method has the advantages of high sensitivity, high accuracy, wide detection range and good repeatability. It is suitable for early warning of Shui Hua outbreaks in cyanobacteria.
【作者单位】：山东省城市供排水水质监测中心;山东大学环境科学与工程学院;
【基金】：住房与城乡建设部国家水体污染控制与治理科技重大专项(2012ZX07404-003) 国家科技惠民工程(2013GS370202) 山东省教育厅泰山学者建设工程专项(ts200640025)
【分类号】：R123.1;R440

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