Zhong Yan, Huang Chengyu, Yin Wenya, Vonk R J, Harmsen H M J
Huaxi School of Public Health, Sichuan University, Chengdu 610041, China.
Wei Sheng Yan Jiu. 2003 May;32(3):232-5.
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 degrees C, the centrifugal speed of samples was 700 g for 2 minutes, and the paraformaldehyde-stocks were not kept for more than 5 months at low temperature (-80 degrees C) before analysis; (2) The method had good stability and could detect the differences of colonic microflora composition between objects at a significant level.
为建立用于分析结肠微生物群的荧光原位杂交（FISH）的最佳实验条件，并分析该技术的稳定性和有效性，从志愿者中收集新鲜粪便样本，使用五种针对特定菌群的16S rRNA靶向寡核苷酸探针，并分析了新鲜粪便样本采集后的保存时间和温度、离心速度和时间、样本多聚甲醛储备液的保存时间对结果的影响。通过CV值评估稳定性和有效性。结果表明：（1）最佳实验条件为新鲜粪便样本在4℃下保存不超过12小时，样本离心速度为700g，离心2分钟，多聚甲醛储备液在低温（-80℃）下分析前保存不超过5个月；（2）该方法具有良好的稳定性，能够在显著水平上检测出不同对象之间结肠微生物群组成的差异。
