实时荧光定量PCR法快速测定水中微囊藻

以铜绿微囊藻（Microcystis aeruginosa）16S rRNA基因片段为靶序列设计一对特异性引物，采用Real-time PCR法，对铜绿微囊藻进行定性、定量检测。试验表明，仅含铜绿微囊藻DNA模板的样品有特异性扩增，扩增产物熔解曲线平稳，峰尖且窄，熔解温度为（87±1）℃。以重组质粒pMD-18T-16S为标准品，检测区间为1．1×102 copies/mL～1．1×108 copies/mL，所得标准曲线符合制备实时定量PCR标准曲线的要求，对标准品进行测定，方法检出限为11 copies/mL。用该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。

A Real-Time PCR Assay for Rapid Detection and Quantification of Microcystis in Water

A real-time polymerase chain reaction （ PCR） assay was designed and evaluated for rapid detec-tion and quantification of the algae Microcystis aeruginosa.A pair of specific primers was designed from the se-quence of 16S region,of which the PCR pecificity was examined compared with Chlorella sp.and Nitzschia sp.. PCR amplifications were detected only from samples which contained Microcystis cells and specific signals were not detected from Chlorella sp..Melting curve was stationary and peak was narrow .Melting temperature was （87 ±1）℃.Based on recombinant plasmid pMD -18T-16S for the standard,the standard curve we got has high linearity and correlation coeficient .Moreover,this assay was in accordance with preparative real-time PCR. Using the developed standard curves ,Microcystis aeruginosa could be quantified in agreement with the quantifica-tion by optical microscopy .