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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,P<0.05)。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。
Objective To establish a fluorescence quantitative PCR method for detecting toxic cyanobacteria in water. Methods Twenty ml pure cultures of algae and 500 ml water samples were collected and filtered through a 0.22 μm membrane filter. The algae were collected for DNA extraction. SYBR Green was used to detect microcystin synthase gene mcy A by real-time fluorescence quantitative PCR. The standard curve established with the template standard and the Ct value was obtained and the copy number of mcy A gene and the concentration of cyanobacteria producing microcystin (MC) in the sample were calculated. Results in the linear range of 13.8 ~ 1.38 × 106 copies / L, the regression equation was y = -3.45 lgx +36.2, r = 0.989 9. The detection limit of the method was 10.7 copies / L, the spiked recoveries ranged from 88.4% to 106.0% and RSD from 3.6% to 4.0%. There was a positive correlation between the method and the conventional microscopy on the detection of laboratory-grown toxin-producing Microcystis aeruginosa (r = 0.994 0, P <0.05). Conclusion The method has high sensitivity, high precision, wide detection range and good repeatability. It is suitable for early warning of cyanobacteria blooms.