期刊
SCIENTIFIC REPORTS
卷 9, 期 -, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/s41598-019-41753-6
关键词
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资金
- Clinical and Translational Science Award (CTSA) from the National Institutes of Health
- JSPS Postdoctoral Fellowships for Research Abroad
- YASUDA Medical Foundation Grants for Research Abroad
- A. Alfred Taubman Medical Research Institute Grand Challenge Award
- National Institutes of Health [CA203542, CA217156, HL128046]
- University of Michigan Fast Forward Gastrointestinal (GI) Innovation Fund
- University of Michigan Comprehensive Cancer Center Support Grant
Stool contains DNA shed from cells of the gastrointestinal (GI) tract and has great potential as a biospecimen for non-invasive, nucleic acid-based detection of GI diseases. Whereas methods for studying faecal microbiome DNA are plentiful, there is a lack of well-characterised procedures for stabilisation, isolation, and quantitative analysis of faecal host DNA. We report an optimised pipeline for faecal host DNA analysis from the point-of-collection to droplet digital PCR (ddPCR) absolute quantification of host-specific gene targets. We evaluated multiple methods for preservation and isolation of host DNA from stool to identify the highest performing methods. To quantify host DNA even if present in partially degraded form, we developed sensitive, human-specific short-amplicon ddPCR assays targeting repetitive nuclear genomic elements (LINE-1) and mitochondrial genes. We validated the ability of these optimised methods to perform absolute quantification of host DNA in 200 stool DNA extracts from samples that were serially collected from three healthy individuals and three hospitalised patients. These specimens allowed assessment of host DNA day-to-day variability in stool specimens with widely varying physical characteristics (i.e., Bristol scores). We further extended this approach to mouse stool analysis, to enable faecal host DNA studies in animal disease models as well.
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