实时荧光定量聚合酶链反应快速检测产毒铜绿微囊藻

摘要

A Real-time polymerase chain reaction(PCR) assay was designed and evaluated for rapid detection and quantification of the toxic algae Microcystis aeruginosa.A pair of specific primers was designed from the sequence of mcyA region,of which the PCR pecificity was examined compared with Chlorella sp..PCR amplifications were detected only from samples which contained M.aeruginosa cells and specific signals were not detected from Chlorella sp..Melting curve was stationary and peak was narrow.Melting temperature was (83 ± 1) ℃.Based on recombinant plasmid pMD-18T-mcyA for the standard,the standard curve we got has high linearity and correlation coefficient.Moreover,this assay was in accordance with preparative qPCR.Using the developed standard curves,M.aeruginosa could be quantified in agreement with the quantifcation by optical microscopy.%以铜绿微囊藻(Microcystis aeruginosa)产毒株微囊藻毒素合成酶基因(mcyA)为靶序列设计了一对特异性引物,运用实时荧光定量聚合酶链反应(qPCR)方法,对铜绿微囊藻进行了定性定量检测.结果表明,仅含钢绿微囊藻脱氧核糖核酸(DNA)模板的样品有扩增,对照组小球藻(Chlorella)没有检测到扩增信号；扩增产物熔解曲线平稳,峰尖且窄,熔解温度为(83±1)℃,证明该聚合酶链反应扩增产物极为特异.以重组质粒pMD-18T-mcyA为标准品,所得标准曲线具有高度线性相关性,重复性好,符合制备实时定量聚合酶链反应标准曲线的要求.利用该方法建立的标准曲线对实验室培养获得的铜绿微囊藻DNA样品进行定量检测,与显微镜计数结果基本一致.