Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis
Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis
复制标题
DOI：
10.1111/j.1574-6968.2012.02523.x
复制
发表时间：
2012-04-01
影响因子：
2.1
通讯作者：
Licht, Tine Rask
中科院分区：
文献类型：
--
作者：
Bahl, Martin Iain;Bergstrom, Anders;Licht, Tine Rask
在DNA提取和下游分析之前冷冻粪便样本广泛用于人类微生物群的宏基因组研究，但可能影响推断的群落组成。在本研究中，采用三种不同的提取方法，对三种均质人类粪便样本进行直接提取或冷冻保存后提取DNA。在新鲜和冷冻样品的提取中，DNA产量没有一致的差异；然而，不同提取方法之间存在差异。利用6对不同引物对重要菌群的16S rRNA基因进行定量PCR分析，通过比较计算丰度的具体比例来评估群落组成。在9个案例中，有7个被冷冻的粪便样本中厚壁菌门和拟杆菌门16S rRNA的基因比例明显高于未被冷冻的相同样本。对这两个门细菌群的qPCR分析进一步支持了这一效应。结果表明，粪便样品的储存条件可能会对测定的厚壁菌门与拟杆菌门比率产生不利影响，该比率是肠道微生物学中常用的生物标志物。
Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology.