Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces.
Author(s): Nechvatal JM, Ram JL, Basson MD, Namprachan P, Niec SR, Badsha KZ, Matherly LH, Majumdar AP, Kato I
Publication: J Microbiol Methods, 2008, Vol. 72, Page 124-32
PubMed ID: 18162191 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of preservation method, duration and temperature of storage, and extraction method on DNA yields and amplificability from fecal specimens. Aliquots from 10 specimens were included in the study. Specimens were collected in plastic containers, placed on ice, and either transferred to RNAlater or PAXgene and stored for 5 days at room temperature, air-dried on Whatman FTA cards or over silica gel beads and stored for 5 days at room temperature, immediately snap frozen in liquid nitrogen, or stored at 4 degrees C for 24 h. After refrigeration, freezing, or room temperature storage, all specimens were stored at -80 degrees C until extraction and analysis.
Summary of Findings:
The authors state that the extraction method accompanying the Whatman FTA cards failed to extract DNA from the fecal specimens and was not included in the study. The authors also report that Nanodrop spectrophotometry results for measuring DNA concentration were frequently variably higher than fluorometric measurements, and so the Picogreen fluorometric method was used. The highest DNA yields were obtained from specimens preserved by RNAlater, PAXgene, or snap-freezing in liquid nitrogen and with extraction by Qiagen Stool Mini. DNA was successfully extracted after fixed or air-dried specimens underwent 5 day room temperature delays and after fresh specimens underwent 24 h 4 degrees C delays before freezing, however, the authors do not examine specimens processed without these delays for comparison. Extraction using Mo-Bio Fecal was clearly inferior, regardless of preservation method. The least PCR inhibition was observed when DNA was obtained from specimens preserved by RNAlater with extraction by Qiagen Stool Mini or preserved by drying on Whatman FTA card with extraction by the modified 2-day Qiagen Mini without lysozyme treatment. The relative proportion of Bacteroides DNA to total DNA extracted was dependent on the preservation and extraction methods, but the proportions from RNAlater-preserved Qiagen Stool-extracted DNA were not significantly different from snap frozen Qiagen Stool-extracted DNA, taken as the control. A PCR targeting hRFC was successful using DNA from all 10 snap frozen Qiagen Stool-extracted specimens and 9 out of 10 RNAlater-preserved Qiagen Stool-extracted specimens after any necessary sample dilution.
Biospecimens
Preservative Types
- Frozen
- RNAlater
- Other Preservative
- PAXgene
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Spectrophotometry DNA Fluorometry DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Air-dried
PAXgene
Refrigeration
RNAlater
Snap frozen
Analyte Extraction and Purification Analyte isolation method Whatman procedure
Mo-Bio Fecal
Qiagen QIAamp DNA Stool Mini
Modified 2-day Qiagen RNA/DNA Mini with lysozyme treatment
Modified 2-day Qiagen RNA/DNA Mini with no lysozyme treatment
Storage Storage duration 24 h
5 d
Storage Storage temperature 4 degrees C
Room temperature
Spectrophotometry Specific Technology platform Fluorometry
PCR Specific Targeted nucleic acid hRFC
Real-time qPCR Specific Targeted nucleic acid Bacteroides DNA