Reference : Method optimization for fecal sample collection and fecal DNA extraction.
Scientific journals : Article
Life sciences : Multidisciplinary, general & others
Method optimization for fecal sample collection and fecal DNA extraction.
Mathay, Conny [> >]
Hamot, Gael [> >]
Henry, Estelle [> >]
Georges, Laura [> >]
Bellora, Camille [> >]
Lebrun, Laura mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) >]
de Witt, Brian [> >]
Ammerlaan, Wim [> >]
Buschart, Anna mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) >]
Wilmes, Paul mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) >]
Betsou, Fay [> >]
Biopreservation and biobanking
Yes (verified by ORBilu)
United States
[en] BACKGROUND: This is the third in a series of publications presenting formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report here optimization of a stool processing protocol validated for fitness-for-purpose in terms of downstream DNA-based analyses. METHODS: Stool collection was initially optimized in terms of sample input quantity and supernatant volume using canine stool. Three DNA extraction methods (PerkinElmer MSM I(R), Norgen Biotek All-In-One(R), MoBio PowerMag(R)) and six collection container types were evaluated with human stool in terms of DNA quantity and quality, DNA yield, and its reproducibility by spectrophotometry, spectrofluorometry, and quantitative PCR, DNA purity, SPUD assay, and 16S rRNA gene sequence-based taxonomic signatures. RESULTS: The optimal MSM I protocol involves a 0.2 g stool sample and 1000 muL supernatant. The MSM I extraction was superior in terms of DNA quantity and quality when compared to the other two methods tested. Optimal results were obtained with plain Sarstedt tubes (without stabilizer, requiring immediate freezing and storage at -20 degrees C or -80 degrees C) and Genotek tubes (with stabilizer and RT storage) in terms of DNA yields (total, human, bacterial, and double-stranded) according to spectrophotometry and spectrofluorometry, with low yield variability and good DNA purity. No inhibitors were identified at 25 ng/muL. The protocol was reproducible in terms of DNA yield among different stool aliquots. CONCLUSIONS: We validated a stool collection method suitable for downstream DNA metagenomic analysis. DNA extraction with the MSM I method using Genotek tubes was considered optimal, with simple logistics in terms of collection and shipment and offers the possibility of automation. Laboratories and biobanks should ensure protocol conditions are systematically recorded in the scope of accreditation.
Luxembourg Centre for Systems Biomedicine (LCSB): Eco-Systems Biology (Wilmes Group)

File(s) associated to this reference

Fulltext file(s):

Limited access
final.pdfPublisher postprint642.24 kBRequest a copy

Bookmark and Share SFX Query

All documents in ORBilu are protected by a user license.