检测微囊藻毒素合成酶基因mcyH的SYBR GreenⅠ实时荧光定量RT-PCR方法的建立

Quantification of microcystin synthetase gene mcyH using real-time quantitative RT-PCR with SYBR greenⅠ

利用RT-PCR方法从铜绿微囊藻PCC7806 mcy基因中扩增出目的基因片段,用TA克隆方法将基因片段插入pGEMT-T Easy中来制备质粒标准品.通过不同连续稀释倍数的质粒标准品成功构建了16srRNA和mcyH两基因的标准曲线,该法构建的两基因标准曲线r2分别为0.9916和0.9938,且质粒标准品具有较好的重复性,满足实时荧光定量RT-PCR的要求.

Microcystis aeruginosa 7806 mcy genes RT- PCR product was subcloned into PGEM-T vector to contrust recombinant plasmid. Standard curves of 16srRNA and mcyH were developped by different folds serial diluted dplasmid DNA, and r^2 of two standard curves separately was 0.9916 and 0. 9938 by the method, The plasmid standard has a better reproducibility. It can satisfy the experiments need.