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

利用基于亚硝酸盐氧化菌(nitrite-oxidizing bacteria,NOB)16S rRNA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.

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 S rRNA sequence of nitrlte-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 NOB consisted of a hybridization temperature of 46℃, hybridization time of 3 hours, and hybridization solution containing 60 mmol·L^-1 NaCl. A specific procedure was established and used to detect the change in abundance of the nitrite- oxidizing bacteria community. Our results suggest FISH technology is a fast and accurate method for quantitative detection of environmental microorganisms.