Ku Hye-Jin, Lee Ju-Hoon
Department of Food Science and Biotechnology, Institute of Life Sciences and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea.
J Microbiol Biotechnol. 2014 Jun 28;24(6):812-22. doi: 10.4014/jmb.1403.03032.
Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new longrange 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias.
对人类肠道微生物群的宏基因组分析扩展了我们对这些细菌在改善人类肠道健康中作用的理解；然而，一些报告表明，目前的总粪便DNA提取方法和16S rRNA通用引物组可能会影响这些分析的物种覆盖范围和分辨率。在此，我们通过优化裂解缓冲液、煮沸时间（10分钟）和珠磨时间（0分钟）改进了从人类粪便样本中提取总DNA的方法。此外，我们开发了一种新的针对V6至V9区域的长片段16S rRNA通用PCR引物组，其DNA产物长度为580 bp。通过与之前开发的两种16S rRNA通用引物组进行比较，对这种新的16S rRNA引物组进行了评估，结果显示其具有高物种覆盖范围和分辨率。优化后的总粪便DNA提取方法和新设计的长片段16S rRNA通用引物组将有助于对成人和婴儿肠道微生物群进行高度准确的宏基因组分析，并将偏差降至最低。
