污染控制技术及原理
张星,林炜铁,朱雅楠.FISH技术定量解析亚硝酸盐氧化菌的条件优化[J].环境科学学报,2009,29(4):716-722
FISH技术定量解析亚硝酸盐氧化菌的条件优化
- Optimization of FISH to detect nitrite-oxidizing bacteria quantitatively
- 基金项目：广东省农业科技攻关计划项目(No.2008B021000036)
- 作者
- 单位
- 张星
- 华南理工大学生物科学与工程学院, 广州 510006
- 林炜铁
- 华南理工大学生物科学与工程学院, 广州 510006
- 朱雅楠
- 华南理工大学生物科学与工程学院, 广州 510006
- 摘要：利用基于亚硝酸盐氧化菌(nitrite-oxidizing bacteria,NOB)16SrRNA序列的荧光探针,优化了针对亚硝酸盐氧化菌的FISH技术实验条件.确定了FISH技术定量检测亚硝酸盐氧化菌的具体方法,并优化了样品预处理的实验条件为热固定2h,4%多聚甲醛固定15min,乙醇脱水5min;杂交过程的实验条件为杂交温度46℃,杂交时间3h,洗脱液中NaCl浓度为60mmol·L-1.运用建立的FISH技术检测了7d内亚硝酸盐氧化菌菌群的丰度变化,充分显示了荧光原位杂交技术(Fluorescence in Situ Hybridization,FISH)的快速定性定量检测优势.
- Abstract：Experimental conditions of Fluorescence in Situ Hybridization(FISH)for detecting nitrite-oxidizing bacteria(NOB)are optimized through orthogonal tests,by using the common fluorescent probe based on the 16 SrRNAsequence of nitrite-oxidizing bacteria.Our results showed that the optimal conditions for sample preparation were a heat fixing-time of 2 hours,4% paraformaldehyde fixing-time of 15 minutes,and ethanol dehydration time of 5 minutes.The optimal conditions for detecting NOBconsisted of a hybridization temperature of 46℃,hybridization time of 3 hours,and hybridization solution containing 60mmol·L-1 NaCl.Aspecific procedure was established and used to detect the change in abundance of the nitrite-oxidizing bacteria community.Our results suggest FISHtechnology is a fast and accurate method for quantitative detection of environmental microorganisms.
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