Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, 3610 Hamilton Walk, Philadelphia, PA, 19104-6076, USA.
Department of Obstetrics and Gynecology, Maternal and Child Health Research Program, Perelman School of Medicine at the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA, 19104, USA.
Microbiome. 2016 Jun 23;4(1):29. doi: 10.1186/s40168-016-0172-3.
Recent studies have suggested that bacteria associated with the placenta-a "placental microbiome"-may be important in reproductive health and disease. However, a challenge in working with specimens with low bacterial biomass, such as placental samples, is that some or all of the bacterial DNA may derive from contamination in dust or commercial reagents. To investigate this, we compared placental samples from healthy deliveries to a matched set of contamination controls, as well as to oral and vaginal samples from the same women.
We quantified total 16S rRNA gene copies using quantitative PCR and found that placental samples and negative controls contained low and indistinguishable copy numbers. Oral and vaginal swab samples, in contrast, showed higher copy numbers. We carried out 16S rRNA gene sequencing and community analysis and found no separation between communities from placental samples and contamination controls, though oral and vaginal samples showed characteristic, distinctive composition. Two different DNA purification methods were compared with similar conclusions, though the composition of the contamination background differed. Authentically present microbiota should yield mostly similar results regardless of the purification method used-this was seen for oral samples, but no placental bacterial lineages were (1) shared between extraction methods, (2) present at >1 % of the total, and (3) present at greater abundance in placental samples than contamination controls.
We conclude that for this sample set, using the methods described, we could not distinguish between placental samples and contamination introduced during DNA purification.
最近的研究表明，与胎盘相关的细菌（“胎盘微生物组”）可能在生殖健康和疾病中起着重要作用。然而，在处理细菌生物量低的标本（如胎盘样本）时，存在一个挑战，即部分或全部细菌 DNA 可能源自灰尘或商业试剂中的污染。为了研究这个问题，我们将健康分娩的胎盘样本与一组匹配的污染对照样本进行了比较，同时还与同一女性的口腔和阴道样本进行了比较。
我们使用定量 PCR 定量了 16S rRNA 基因的总拷贝数，发现胎盘样本和阴性对照中含有低且无法区分的拷贝数。相比之下，口腔和阴道拭子样本的拷贝数较高。我们进行了 16S rRNA 基因测序和群落分析，发现胎盘样本和污染对照的群落之间没有分离，尽管口腔和阴道样本显示出特征性的、独特的组成。两种不同的 DNA 纯化方法得到了类似的结论，尽管污染背景的组成不同。真实存在的微生物群应该产生类似的结果，无论使用哪种纯化方法——这在口腔样本中是可见的，但没有一种胎盘细菌谱系（1）在两种提取方法之间共享，（2）在总样本中存在超过 1%，并且（3）在胎盘样本中的丰度高于污染对照。
我们的结论是，对于这个样本集，使用描述的方法，我们无法区分胎盘样本和 DNA 纯化过程中引入的污染。
