Highly sensitive real-time PCR assay for quantification of toxic cyanobacteria based on microcystin synthetase a gene
Highly sensitive real-time PCR assay for quantification of toxic cyanobacteria based on microcystin synthetase a gene
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DOI：
10.1263/jbb.102.90
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发表时间：
2006-08-01
影响因子：
2.8
通讯作者：
Inamori, Yuhei
中科院分区：
文献类型：
--
作者：
Furukawa, Kazuhiro;Noda, Naohiro;Inamori, Yuhei
供水水库中出现蓝藻水华可能对健康造成潜在危害。在这项研究中，我们的目的是定量的微囊藻毒素产生的蓝藻微囊藻毒素合成酶A（mcyA）基因的基础上，使用实时PCR。为了实现高灵敏度的实时荧光定量PCR检测，设计了一种新的引物MSR-2 R，并采用共沉淀法提取DNA。蓝藻细胞可以有效地收集悬浮在溶液中的其他细菌的共沉淀，即使在低浓度的蓝藻的情况下。发现该方法的检测限为8.8个细胞/反应。当在纯培养中监测蓝藻生长时，通过实时PCR确定的细胞浓度与从直接显微镜计数确定的细胞浓度正相关。此外，我们还可以使用实时PCR检测和定量湖泊水样中的mcyA基因。结果表明，基于实时荧光定量PCR技术的mcyA基因的定量检测方法是一种快速检测环境样品中产微囊藻毒素蓝藻的有效方法。
The presence of cyanobacterial bloom in water supply reservoirs can cause potential health hazards. In this study, we aimed at the quantification of microcystin-producing cyanobacteria based on the microcystin synthetase A (mcyA) gene using real-time PCR. To perform a highly sensitive real-time PCR assay, the novel primer MSR-2R was designed and a coprecipitation DNA extraction method was used in this study. Cyanobacterial cells could be collected efficiently by coprecipitation with other bacteria suspended in solution even in the case of low concentrations of cyanobacteria. The detection limit of the method was found to be 8.8 cells per reaction. When cyanobacterial growth was monitored in pure culture, the cell concentration determined by real-time PCR positively correlated with the cell concentration determined from direct microscopic count. Furthermore, we could detect and quantify the mcyA gene in lake water samples using real-time PCR. It was concluded that the quantification of the mcyA gene based on real-time PCR is a powerful tool for the rapid quantification of microcystin-producing cyanobacteria in environmental samples.