应用生态学报2013，Vol.24Issue(2)：541-548,8.
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
摘要
Abstract
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 μX 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.关键词
东海原甲藻/定量PCR/细胞色素b基因/内参基因Key words
Prorocentrum donghaiense Lu/ real-time fluorescent quantitative PCR ( RFQ-PCR) /cytochrome b/ reference gene分类
生物科学引用本文复制引用
王曲圆,甄毓,袁健,米铁柱,于志刚..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[J].应用生态学报,2013,24(2):541-548,8.基金项目
国家重点基础研究发展计划项目(2011CB403602)和海洋公益性行业科研专项(201105014-03,201205031-03)资助. （2011CB403602）
