Nechvatal Jordan M, Ram Jeffrey L, Basson Marc D, Namprachan Phanramphoei, Niec Stephanie R, Badsha Kawsar Z, Matherly Larry H, Majumdar Adhip P N, Kato Ikuko
Department of Physiology, Wayne State University, 540 E. Canfield Avenue, Detroit, MI 48201, USA.
J Microbiol Methods. 2008 Feb;72(2):124-32. doi: 10.1016/j.mimet.2007.11.007. Epub 2007 Nov 21.
Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastrointestinal tract health. Intestinal bacteria that synthesize or metabolize potential carcinogens and produce anti-tumorigenic products may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. To facilitate epidemiological studies relating bacterial and epithelial cell DNA and RNA markers, preservative/extraction methods suitable for self-collection and shipping of fecal samples at room temperature were tested. Purification and PCR amplification of fecal DNA were compared after preservation of stool samples in RNAlater (R) or Paxgene (P), or after drying over silica gel (S) or on Whatman FTA cards (W). Comparisons were made to samples frozen in liquid nitrogen (N2). DNA purification methods included Whatman (accompanying FTA cards), Mo-Bio Fecal (MB), Qiagen Stool (QS), and others. Extraction methods were compared for amount of DNA extracted, DNA amplifiable in a real-time SYBR-Green quantitative PCR format, and the presence of PCR inhibitors. DNA can be extracted after room temperature storage for five days from W, R, S and P, and from N2 frozen samples. High amounts of total DNA and PCR-amplifiable Bacteroides spp. DNA (34%+/-9% of total DNA) with relatively little PCR inhibition were especially obtained with QS extraction applied to R preserved samples (method QS-R). DNA for human reduced folate carrier (SLC19A1) genomic sequence was also detected in 90% of the QS-R extracts. Thus, fecal DNA is well preserved by methods suitable for self-collection that may be useful in future molecular epidemiological studies of intestinal bacteria and human cancer markers.
粪便中含有肠道细菌和脱落的上皮细胞，这些可能提供有关胃肠道健康的有用信息。合成或代谢潜在致癌物并产生抗肿瘤产物的肠道细菌可能与结直肠癌有关，结直肠癌是美国癌症死亡的第二大常见原因。为了促进与细菌和上皮细胞DNA及RNA标记相关的流行病学研究，测试了适用于在室温下自行采集和运输粪便样本的防腐剂/提取方法。比较了将粪便样本保存在RNA Later（R）或Paxgene（P）中，或在硅胶（S）上或Whatman FTA卡（W）上干燥后粪便DNA的纯化和PCR扩增情况。并与液氮（N2）冷冻的样本进行了比较。DNA纯化方法包括Whatman（随附FTA卡）、Mo-Bio粪便（MB）、Qiagen粪便（QS）等。比较了不同提取方法提取的DNA量、在实时SYBR-Green定量PCR格式中可扩增的DNA以及PCR抑制剂的存在情况。从W、R、S和P以及N2冷冻样本中室温保存五天后均可提取DNA。特别是应用QS提取法处理保存在R中的样本（方法QS-R）时，可获得大量的总DNA和可PCR扩增的拟杆菌属DNA（占总DNA的34%±9%），且PCR抑制相对较少。在90%的QS-R提取物中还检测到了人类还原叶酸载体（SLC19A1）基因组序列的DNA。因此，通过适用于自行采集的方法可以很好地保存粪便DNA，这可能对未来肠道细菌和人类癌症标记的分子流行病学研究有用。
