东海原甲藻细胞色素b基因实时荧光定量PCR检测体系优化

Optimization of real-time quantitative fluorescence PCR system in detecting cytochrome b gene of Prorocentrum donghaiense Lu

为定量检测现场样品中东海原甲藻的细胞色素b(cytochromeb,Cytb)基因,本研究设计了该基因的特异性引物,并对现场样品反转录反应体系中加入的模板数量和定量PCR反应条件进行了优化.结果表明:设计的引物具有较好的特异性,可有效区分不同的藻类;针对现场样品,在20μL的反转录体系中,适宜加入的RNA模板的量为50～200ng;PCR模板稀释10倍或向定量PCR反应体系中加入终浓度为0.2μg·μL-1的牛血清蛋白(BSA)均能有效降低现场样品中抑制物的抑制作用,减小干扰.该方法的建立对从分子水平探讨东海原甲藻暴发和消亡的内在机制具有重要意义.

To quantitatively detect the cytochrome b（Cyt b） gene of Prorocentrum donghaiense Lu in the filed samples,a specific primer was designed,and the quantities of the RNA templates added into the reaction system for reverse transcription as well as the reaction conditions of real-time fluorescent quantitative PCR（RFQ-PCR） were optimized.The results illustrated that the designed primer had good specificity,being able to be used to differentiate different algal species effectively.In detecting the filed samples,the suitable qualities of the templates for the 20 μL reverse transcription system were 50-200 ng.10-fold diluting the templates or adding the bovine serum albumin（BSA） with a final concentration 0.2 μg·μL-1 into the RFQ-PCR system could effectively decrease the inhibitory effect of the inhibitors in the filed samples,and thus,decrease the interferences.The established real-time fluorescent quantitative PCR（RFQ-PCR） assay would facilitate us to study the intrinsic mechanisms of P.donghaiense outbreak and extinction at molecular level.