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

应用生态学报

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

王曲圆¹,甄毓²,袁健¹,米铁柱^2**,于志刚³

(¹中国海洋大学海洋生命学院，山东青岛 266003； ^2 中国海洋大学环境科学与工程学院海洋环境与生态教育部重点实验室，山东青岛 266100； ^3中国海洋大学化学化工学院海洋化学理论与工程技术教育部重点实验室，山东青岛 266100)

出版日期:2013-02-18 发布日期:2013-02-18

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

WANG Qu-yuan¹, ZHEN Yu², YUAN Jian¹, MI Tie-zhu², YU Zhi-gang³

(¹College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong, China; ²Ministry of Education Key Laboratory of Marine Environment & Ecology, College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, Shandong, China; ^3Ministry of Education Key Laboratory of Marine Chemistry Theory & Technology, College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, Shandong, China)

Online:2013-02-18 Published:2013-02-18

摘要/Abstract

摘要： 为定量检测现场样品中东海原甲藻的细胞色素b(cytochrome b, Cyt b)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20 μL的反转录体系中，适宜加入的RNA模板的量为50~200 ng；PCR模板稀释10倍或向定量PCR反应体系中加入终浓度为0.2 μg·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.

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 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.

王曲圆1,甄毓2,袁健1,米铁柱2**,于志刚3. 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.

WANG Qu-yuan1, ZHEN Yu2, YUAN Jian1, MI Tie-zhu2, YU Zhi-gang3. Optimization of real-time quantitative fluorescence PCR system in detecting cytochrome b gene of Prorocentrum donghaiense Lu.[J]. Chinese Journal of Applied Ecology.