Micribiology
Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________
Comparison of methods for the extraction of bacterial DNA from human faecal samples for analysis by real-time PCR
E.A Nelson1, 2, E.A Palombo1, and S.R Knowles2
1
Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology, John Street Hawthorn, VIC 3122, Australia. 2 SwinPsyche Unit, Faculty of Life and Social Sciences, Swinburne University of Technology, John Street Hawthorn, VIC 3122, Australia. Real-time PCR analysis of bacterial DNA isolated from faecal specimens has become increasingly used for the quantification of indigenous intestinal microbiota. The success of such analysis requires effective methods for the extraction of faecal bacterial DNA. Three extraction methods were assessed for their effectiveness in extracting Escherichia coli and Enterococcus spp. bacterial DNA from human faecal samples: an in-house phenol/chloroform extraction, and two commercially available kits, ExtractMaster (Epicentre Biotechnologies) and UltraClean (Mo Bio Laboratories) faecal DNA extraction kits. Real-time PCR using the standard curve method was used to quantify the level of bacterial DNA extracted from ten faecal samples. The phenol/chloroform method required an additional dilution step before DNA could be amplified by real-time PCR. After taking into account this dilution, the three extraction methods did not differ in the level of E. coli bacterial DNA detected. However, for Enterococcus, the ExtractMaster kit resulted in significantly less DNA detected. The phenol/chloroform method reliably extracted DNA, and produced extracts with short-term stability. Extraction using phenol/chloroform produces quantities of faecal bacterial DNA comparable to commercially available kits when amplified by current
Comparison of methods for the extraction of bacterial DNA from human faecal samples for analysis by real-time PCR
E.A Nelson1, 2, E.A Palombo1, and S.R Knowles2
1
Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology, John Street Hawthorn, VIC 3122, Australia. 2 SwinPsyche Unit, Faculty of Life and Social Sciences, Swinburne University of Technology, John Street Hawthorn, VIC 3122, Australia. Real-time PCR analysis of bacterial DNA isolated from faecal specimens has become increasingly used for the quantification of indigenous intestinal microbiota. The success of such analysis requires effective methods for the extraction of faecal bacterial DNA. Three extraction methods were assessed for their effectiveness in extracting Escherichia coli and Enterococcus spp. bacterial DNA from human faecal samples: an in-house phenol/chloroform extraction, and two commercially available kits, ExtractMaster (Epicentre Biotechnologies) and UltraClean (Mo Bio Laboratories) faecal DNA extraction kits. Real-time PCR using the standard curve method was used to quantify the level of bacterial DNA extracted from ten faecal samples. The phenol/chloroform method required an additional dilution step before DNA could be amplified by real-time PCR. After taking into account this dilution, the three extraction methods did not differ in the level of E. coli bacterial DNA detected. However, for Enterococcus, the ExtractMaster kit resulted in significantly less DNA detected. The phenol/chloroform method reliably extracted DNA, and produced extracts with short-term stability. Extraction using phenol/chloroform produces quantities of faecal bacterial DNA comparable to commercially available kits when amplified by current
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