中文摘要：

根据博德特氏菌（Bordetella sp．）的16SrRNA基因序列，设计荧光原位杂交（FISH）检测博德特氏菌的寡核苷酸探针FW_iso_62和Fw_iso_761，存20％～60％甲酰胺均有很强的荧光信号。采用探针FW_iso_62及其竞争探针，结合Nycodenz和DAPI技术，建立定量检测土壤中博德特氏菌的DAPI—FISH方法。该方法可排除土壤颗粒的自动荧光对细菌信号的掩盖，保证图片中有大量微生物供统计分析，还能有效保存微生物的原位信息。应用该方法分析土壤中1，2，4-三氯苯降解菌-博德特氏菌，结果未受氯苯污染的农田土壤中没有检测到博德特氏菌，而氯苯污染土壤巾检测到大量的博德特氏菌，每克土壤含3.78×10^6个。将该污染土壤中分离的博德特氏降解菌及其降斛菌群接种至农田土壤中，降解菌的数量均随培养而增加，个月后分别占DAPI计数的1．7％和3．8％。本研究设计的探针可有效用于复杂环境样品中博德特氏菌的定性与定最检测，

英文摘要：

Novel 16S-rRNA targeted oligonucleotide probes （FW iso 62 and FW iso 761） were designed to detect Bordetella sp. by fluorescence in situ hybridization （FISH）. Strong fluorescence signals with the probes were observed in 20% - 60% formamide concentrations. Quantification of Bordetella sp. in soil was developed with the selected probe of FW iso 62 and its competitor combined with Nycodenz and DAPI technique. The advantages of the established DAPI- FISH ： eliminate autofluorescence of soil particles masking the bacterial signals; ensure a large amount of bacteria in one picture for reliable statistical analysis ; and keep efficiently in situ information of the bacteria. 1,2,4-trichlorobenzene de- grading Bordetella bacteria were investigated in soils with the established method. The Bordetella bacterium was not detected in the agricultural soil, which was not exposed to chlorobenzenes. On the contrary, a lot of Bordetella bacteria （3.78 × 10^6 cell g^-1） were determined in the soil contaminated with chlorobenzenes. When the 1,2,4-TCB-degrading Bordetella strain and its community in the contaminated soil were re-inoculated into the agricultural soil, the Bordetella numbers increased and accounted for 1.7% and 3.8% of the DAPI counts after incubation for 1 month, respectively. The probes developed in this study are useful for detecting Bordetella bacteria in complicated environmental samples.