Culture independent DNA extraction method for bacterial cells concentrated from water
• 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...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-01-01
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| Series: | MethodsX |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2215016122000371 |
| _version_ | 1811178828491390976 |
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| author | K.B. Hoorzook T.G. Barnard |
| author_facet | K.B. Hoorzook T.G. Barnard |
| author_sort | K.B. Hoorzook |
| collection | DOAJ |
| description | • 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. |
| first_indexed | 2024-04-11T06:25:30Z |
| format | Article |
| id | doaj.art-bb14674e34db48499d10847e562b06fe |
| institution | Directory Open Access Journal |
| issn | 2215-0161 |
| language | English |
| last_indexed | 2024-04-11T06:25:30Z |
| publishDate | 2022-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | MethodsX |
| spelling | doaj.art-bb14674e34db48499d10847e562b06fe2022-12-22T04:40:25ZengElsevierMethodsX2215-01612022-01-019101653Culture independent DNA extraction method for bacterial cells concentrated from waterK.B. Hoorzook0T.G. Barnard1Corresponding author.; University of Johannesburg, South AfricaUniversity of Johannesburg, South Africa• 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.http://www.sciencedirect.com/science/article/pii/S2215016122000371Culture independent DNA extraction method |
| spellingShingle | K.B. Hoorzook T.G. Barnard Culture independent DNA extraction method for bacterial cells concentrated from water MethodsX Culture independent DNA extraction method |
| title | Culture independent DNA extraction method for bacterial cells concentrated from water |
| title_full | Culture independent DNA extraction method for bacterial cells concentrated from water |
| title_fullStr | Culture independent DNA extraction method for bacterial cells concentrated from water |
| title_full_unstemmed | Culture independent DNA extraction method for bacterial cells concentrated from water |
| title_short | Culture independent DNA extraction method for bacterial cells concentrated from water |
| title_sort | culture independent dna extraction method for bacterial cells concentrated from water |
| topic | Culture independent DNA extraction method |
| url | http://www.sciencedirect.com/science/article/pii/S2215016122000371 |
| work_keys_str_mv | AT kbhoorzook cultureindependentdnaextractionmethodforbacterialcellsconcentratedfromwater AT tgbarnard cultureindependentdnaextractionmethodforbacterialcellsconcentratedfromwater |