Culture independent DNA extraction method for bacterial cells concentrated from water

K. B. Hoorzook, T. G. Barnard

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

• To bypass the problem of viable but non-culturable bacteria that cannot be isolated by culturable methods would be to isolate DNA from bacterial cells concentrated from water samples used as a template for the polymerase chain reactions (PCR). DNA extraction protocol (Omar et al. 2010) was used as a foundation for extracting Escherichia coli DNA from water. The method combinations i.e., guanidium thiocyanate, celite and home-made spin column were chosen because it has been shown to be reliable, rapid, simple, and inexpensive for routine analysis in developing country settings. • The following optimizations were included: (a) Polycarbonate (Poly) was statistically compared with Polyether sulphone (PES), Nitrocellulose acetate (NA) and Nitrocellulose (NC) membranes; (b) Various housing containers for the membranes were tested: plastic/glass petri-dish, Falcon tubes, Ogreiner cryovials; (c) various solutions was tested to add to the membrane to remove cells from membranes; (d) celite was chosen to bind the DNA because it had a higher DNA binding capacity compared to silicon dioxide; (e) incubation times and rotation speed were tested when adding reagents. • The optimized in-house DNA extraction method was validated with environmental water samples, high (dam water) and low (borehole) bacterial load to determine upper and lower detection limits of the method.

Original languageEnglish
Article number101653
JournalMethodsX
Volume9
DOIs
Publication statusPublished - Jan 2022

Keywords

  • Celite
  • Culture independent DNA extraction method
  • Environmental water
  • Escherichia coli
  • Genomic and plasmid extraction
  • Guanidium thiocyanate
  • Membrane filtration

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Medical Laboratory Technology

Fingerprint

Dive into the research topics of 'Culture independent DNA extraction method for bacterial cells concentrated from water'. Together they form a unique fingerprint.

Cite this