Wang Qu-Yuan, Zhen Yu, Yuan Jian, Mi Tie-Zhu, Yu Zhi-Gang
Ying Yong Sheng Tai Xue Bao. 2013 Feb;24(2):541-8.
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 microL 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 microg.microL-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.
为了定量检测野外样本中东海原甲藻（Prorocentrum donghaiense Lu）的细胞色素b（Cyt b）基因，设计了特异性引物，并对反转录反应体系中添加的RNA模板量以及实时荧光定量PCR（RFQ-PCR）的反应条件进行了优化。结果表明，所设计的引物具有良好的特异性，能够有效区分不同藻类物种。在检测野外样本时，20 μL反转录体系中合适的模板量为50 - 200 ng。将模板10倍稀释或向RFQ-PCR体系中添加终浓度为0.2 μg·μL-1的牛血清白蛋白（BSA），可有效降低野外样本中抑制剂的抑制作用，从而减少干扰。所建立的实时荧光定量PCR（RFQ-PCR）检测方法将有助于我们在分子水平上研究东海原甲藻爆发和消亡的内在机制。
