全细胞多重PCR检测蓝藻、微囊藻及产毒微囊藻方法初探

选取三对分别针对微囊藻、蓝藻16S rDNA及微囊藻毒素合成酶基因mcyB的保守序列的特异性引物209F/409R、27F1/409R、MTR/MTF,其中409R为一条共用引物。设计并优化了一种可以同时检测蓝藻和微囊藻的两重全细胞PCR方法和一种可以同时检测蓝藻、微囊藻和可产毒微囊藻的三重全细胞PCR方法,并且测试了这两种PCR反应的灵敏度区间,分别为10⁵～10³cell·mL^-1、10⁵～10²cell·mL^-1。对采集水库水样检测结果表明双重全细胞PCR方法可以直接应用于对天然水样的检测,三重全细胞PCR方法可用于实验室培养藻细胞的筛查。全细胞多重PCR方法具有快速、简便、准确等特点,在水体微囊藻毒素检测预警方面具有应用价值。

Primary studies on the detection of Microcystis, cyanobacteria and microcystin synthetase gene by the whole-cell multiplex PCR

Based on the conserved 16S rDNA sequence of Microcystis, Cyanobacteria and microcystin synthetase gene B(mcyB), three pairs of specific primer 209F/409R, 27F1/409R and MTR/MTF are selected. The primer 409R is shared in two pairs. A duplex PCR to detect Microcystis and Cyanobacteria and a triplex PCR to detect Microcystis, Cyanobacteria and mcyB are optimized. The threshold of primers for cell concentration is also studied to detect the sensitivity of these primers. Results showed that the optimal concentrations for duplex PCR and for triplex PCR are from 10⁵ cells·mL^-1 to 10³ cells·mL^-1, and the duplex PCR could be used directly to detect crude water samples from reservoirs. This study suggested that the multiplex PCR is a simple and practical approach, and could play an important role on monitoring of microcystin in water.