荧光原位杂交技术分析人结肠菌群方法研究

Studies on fluorescence in situ hybridization with group-specific 16S rRAN-based probes in analysis of human colonic microflora

建立荧光原位杂交技术分析人体内结肠菌群的方法。取受试者新鲜粪便 ,选用 5种特异性的 16SrRNA寡核苷酸探针 ,检测粪便样本收集后的保存时间、温度 ,离心条件及样本固定液存放时间对杂交计数结果的影响。结果建立最佳实验条件为 :粪便样本收集后应尽快在 4℃下保存 ,放置时间不要超过 12小时即作处理 ;样本的适宜离心条件为 70 0g 2分钟 ;样本用多聚甲醛固定后在 - 80℃下存放时间不要超过 5个月。该方法具有较好的稳定性 ,可以有效地检出个体之间结肠菌群的差异。

In order to establish the optional experimental conditions of fluorescence in situ hybridization (FISH) for analysis of colonic microflora, and analyze the stability and validation of the technique, fresh fecal samples were collected from the volunteers and five group-specific 16S rRNA-targeted oligonucleotide probes were used, and the effects of keeping time and temperature of fresh fecal samples after collected, the centrifugal speed and time, the storage time of paraformaldehyde-stocks of samples on the results were analyzed. The stability and the validation were assessed by CV values. Results showed that: (1) The optional experimental conditions were that the fresh fecal samples were not kept for more than 12 hours at 4℃, the centrifugal speed of samples was 700g for 2 minutes, and the paraformaldehyde-stocks were not kept for more than 5 mooths at low temperature (-80℃) before analysis; (2) The method had good stability and could detect the differences of colonic microflora composition between objects at a significant level.