PCR-DGGE技术分析浸矿体系细菌群落结构的实验优化  

PCR-DGGE Experimental Condition Optimization in Analysis of Bioleaching Bacterial Community Structure

作　　者：林连兵[1] 张兰兰[1] 丁丽娜[1] 季秀玲[1] 魏云林[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,云南昆明650500

出　　处：《昆明理工大学学报（自然科学版）》2012年第4期14-18,共5页Journal of Kunming University of Science and Technology(Natural Science)

基　　金：国家自然科学基金资助(项目编号:31160035;30960022)

摘　　要：为了研究低品位黄铜矿细菌搅拌浸出的浸矿菌液中的细菌群落结构,对PCR-DGGE(变性梯度凝胶电泳)技术相关的实验方法进行了优化,主要涉及低丰度基因组DNA提取方法优化、16S rRNA基因的PCR扩增条件优化以及变性梯度凝胶电泳条件的优化.结果表明,采用分级离心多次浓缩的方法收集矿浆样品中的菌体,再用试剂盒提取基因组DNA,有较好效果;使用含特定"GC夹板"的引物5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG-3'进行热启动降落PCR,能显著提高目的条带的PCR扩增效率;当DGGE电泳设置温度为60℃左右,胶浓度为6%,尿素变性梯度为30%～50%,恒定电压为100 V,时间为9 h时,可以获得效果较好的DGGE电泳图谱,该优化方案提高了对浸矿体系中细菌群落结构的检测效果.To investigate the bacterial community structure in agitation bioleaching of low - grade chalcopyrite, related experimental condition of PCR - DGGE （ Denaturing Gradient Gel Electrophoresis） technique is optimized. The optimization mainly includes genomic DNA from low -abundance bacterial ore pulp, PCR amplification of 16S rRNA gene and DGGE experimental conditions. The results show that using fractional centrifugation and repeated concentration to collect bacteria in the ore pulp and followed by extraction DNA with Qiagen DNA extraction kit can obtain high quantity genomic DNA. Using special primer containing GC -clamp （5＇ -CGCCCGCCGCGC- CCCGCGCCCGTCCCGCCGCCCCCGCCCG - 3＇） and hot - start PCR amplification procedure can enhance the PCR amplification efficiency. The optimal conditions found for DGGE include 6% polyacrylamide for the gel, a denaturing gradient of 30 - 50% between the lowest and the highest concentration of denaturant, a running time of 9 hours at 100 Volt and keeping the electrophoresis buffer at a temperature of 60℃. The optimization program improves the detective efficiency of bacterial community structure in leaching system.

关 键 词：浸矿体系 16SRRNA基因 PCR-DGGE实验优化 

分 类 号：TF18[冶金工程—冶金物理化学]