基于NGS方法评估不同保存条件对粪便样本中菌群含量的影响

Impact of temperature and time of storage on bacteria in fecal samples for metagenomic analyses

目的评估粪便样本的不同保存条件对肠道微生态研究结果的影响。方法设计相关实验,比较7种不同的保存方法,基于高通量测序技术比对不同时间、不同贮存温度对粪便样本DNA质量、菌群多样性及病原菌检出等结果之间的差异。结果证实了对于粪便样本采集仍建议采取即刻提取核酸或-20℃保存的方法。同时比较多种保存方法后发现,采用不同样本保存方法受到影响的菌属集中在低丰度菌属(≤0.01%),对应较高丰度的病原菌检测结果的可靠性影响较小,样本中丰度超过千分之一的病原菌检测率超过95%。结论对于肠道微生态研究建议对粪便样本采取即刻提取核酸或-20℃保存的方法。对于由于其他原因未能妥善保存的粪便样本(如常温保存48 h),仍可对丰度超过千分之一的病原菌进行检测。以上结果对于肠道菌群研究中异地所采集的粪便样本运输和贮存具有一定的指导意义。

Objective To evaluate the effect of storage temperature and time on DNA quality.Methods Correlation experiments were designed to compare 7 different storage methods.Based on high-throughput sequencing technology,the differences in DNA quality,bacterial diversity and pathogen detection of fecal samples were compared between different storage temperatures and time.Results The method of immediate extraction of nucleic acid and storage temperature of-20℃were best for fecal sample collection.Different storage methods did not affect the reliability of detection results of pathogens with higher abundances,and the detection rates of pathogens with abundance over one thousandth were higher than 95%.Conclusion For the study of intestinal microecology,it is recommended to immediately extract nucleic acid from fecal samples or to preserve them at-20℃.For fecal samples that have not been properly stored for other reasons(e.g.,48 h at room temperature),the detection of pathogenic bacteria with abundance greater than 1/1000 can still be carried out.