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DNA提取和脂质去除对人乳细菌谱的影响。

The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles.

作者信息

Ojo-Okunola Anna, Claassen-Weitz Shantelle, Mwaikono Kilaza S, Gardner-Lubbe Sugnet, Zar Heather J, Nicol Mark P, du Toit Elloise

机构信息

Division of Medical Microbiology, Department of Pathology, Faculty of Health Sciences, Observatory 7925, University of Cape Town, Cape Town 7700, South Africa.

Computational Biology Group and H3ABioNet, Department of Integrative Biomedical Sciences, Observatory 7925, University of Cape Town, Cape Town 7700, South Africa.

出版信息

Methods Protoc. 2020 May 15;3(2):39. doi: 10.3390/mps3020039.

DOI:10.3390/mps3020039
PMID:32429170
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7359716/
Abstract

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of and . DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.

摘要

不依赖培养的分子技术推动了对环境样本和人体样本（包括人乳（HM）细菌群落）的特征描述。然而，提取能够代表样本中细菌群体的高质量基因组DNA至关重要。在DNA提取之前从人乳中去除脂质是常见做法，但这可能会影响检测到的细菌群体。本研究的目的是比较四种商业DNA提取试剂盒以及与母乳细菌谱相关的脂质去除情况。使用从十名健康哺乳期妇女收集的乳汁评估了四种商业DNA提取试剂盒，即QIAamp DNA Microbiome试剂盒、ZR Fungal/Bacterial DNA MiniPrep™、QIAsymphony DSP DNA试剂盒和ZymoBIOMICS™ DNA Miniprep试剂盒。这些试剂盒根据从人乳中提取大量纯DNA的能力以及从添加到母乳中的商业模拟微生物群落标准中存在的细菌群落提取DNA的效果进行评估。最后，通过评估试剂盒的提取重复性对其进行评价。使用Illumina MiSeq测序靶向16S rRNA基因的V4区域评估细菌谱。ZR Fungal/Bacterial DNA MiniPrep™和ZymoBIOMICS™ DNA Miniprep（美国加利福尼亚州欧文市的Zymo Research公司）试剂盒提取的DNA产量最高且纯度最佳。使用ZR Fungal/Bacterial DNA MiniPrep™提取的DNA最能代表添加到母乳中的模拟群落中的细菌。在未添加模拟群落的母乳样本中，使用QIAsymphony DSP DNA试剂盒提取的DNA在分类群患病率方面与使用ZR Fungal/Bacterial DNA MiniPrep™和ZymoBIOMICS™ DNA Miniprep试剂盒提取的DNA存在统计学上的显著差异。脱脂牛奶和全脂牛奶之间的唯一差异体现在具有不同相对丰度的 和 的细菌谱中。DNA提取而非脂质去除会显著影响在母乳样本中检测到的细菌谱，这强调了仔细选择DNA提取试剂盒以提高从一系列细菌分类群中回收DNA的必要性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4478/7359716/520ca48fd4b7/mps-03-00039-g007.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4478/7359716/d6456ff9160d/mps-03-00039-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4478/7359716/3aa7669b9196/mps-03-00039-g003.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4478/7359716/520ca48fd4b7/mps-03-00039-g007.jpg

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