Optimizing bacterial DNA extraction in urine


Autoři: Matthew M. Munch aff001;  Laura C. Chambers aff002;  Lisa E. Manhart aff002;  Dan Domogala aff001;  Anthony Lopez aff001;  David N. Fredricks aff001;  Sujatha Srinivasan aff001
Působiště autorů: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America aff001;  Department of Epidemiology, University of Washington, Seattle, Washington, United states of America aff002;  Department of Global Health, University of Washington, Seattle, Washington, United states of America aff003;  Department of Medicine, University of Washington, Seattle, Washington, United States of America aff004;  Department of Microbiology, University of Washington, Seattle, Washington, United states of America aff005
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222962

Souhrn

Urine is an acceptable, non-invasive sample for investigating the human urogenital microbiota and for the diagnosis of sexually transmitted infections. However, low quantities of bacterial DNA and PCR inhibitors in urine may prevent efficient PCR amplification for molecular detection of bacteria. Furthermore, cold temperatures used to preserve DNA and bacteria in urine can promote precipitation of crystals that interfere with DNA extraction. Saline, Dulbecco’s Phosphate Buffered Saline, or Tris-EDTA buffer were added to urine from adult men to determine if crystal precipitation could be reversed without heating samples beyond ambient temperature. Total bacterial DNA concentrations and PCR inhibition were measured using quantitative PCR assays to compare DNA yields with and without buffer addition. Dissolution of crystals with Tris-EDTA prior to urine centrifugation was most effective in increasing bacterial DNA recovery and reducing PCR inhibition. DNA recovery using Tris-EDTA was further tested by spiking urine with DNA from bacterial isolates and median concentrations of Lactobacillus jensenii and Escherichia coli 16S rRNA gene copies were found to be higher in urine processed with Tris-EDTA. Maximizing bacterial DNA yield from urine may facilitate more accurate assessment of bacterial populations and increase detection of specific bacteria in the genital tract.

Klíčová slova:

Bacteria – DNA – DNA extraction – Oxalates – Polymerase chain reaction – Ribosomal RNA – Urine – Crystals


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2019 Číslo 9

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