摘要目的 建立微囊藻基因实时荧光定量聚合酶链式反应检测方法,实现微囊藻毒素预警性监测.方法 针对微囊藻毒素合成酶基Mcy A、Mcy D和微囊藻特异性的16S rDNA部分核苷酸序列设计合成引物,将扩增获得的目的基因与pMD@18-T Vector连接制备微囊藻毒素合成酶系编码基因重组质粒标准品,确定检测体系主要参数,构建标准曲线,并据此计算待测水样微囊藻毒素合成酶基因的拷贝数.结果 初始模板量的对数值与Ct值呈良好的线性关系,相关系数r2均达0.995以上,重复性较好.结论 所建立的微囊藻基因荧光定量PCR检测方法,准确度高,灵敏度好,抗干扰性好,实用性强,可用于水源中产毒微囊藻的预测性检测.

abstractsObjective To develop a real-time fluorescent quantitative PCR method for the detection of toxic gene copy number of microcystin algae,so as to realize warning detection of microcystin algae.Methods With the primers based on microcystin synthetase gene mcy A,racy D and partial Microcystis-specific 16S rDNA,the target gene was inserted into pMD (R) 18-T Vector for the preparation of plasmid standards to establish the standard curve of real-time fluorescent quantitative PCR method and through the standard curve to calculate the toxin-producing gene copy number of the unknown sample.Results The exponential curve of the initial template was linear with the Ct value and the related coefficient r2 was all higher than 0.995 and had good reproducibility.The established method could yield the same result as the recommended international standard method.Conclusions With good accuracy,sensitivity,specificity and practicability,this method could be used for predictive detection of water-borne toxin-produced microcystin algae.