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Culture independent DNA extraction method for bacterial cells concentrated from water. | LitMetric

•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 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.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8933712PMC
http://dx.doi.org/10.1016/j.mex.2022.101653DOI Listing

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