Micribiology

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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 real-time PCR technology. Furthermore, phenol/chloroform extraction is rapid and simple, and provides a clear cost advantage for laboratories with limited funds. Keywords DNA extraction; Faecal samples; Real-time PCR

1. Introduction
It is well established that the indigenous microbiota that inhabit the human gastrointestinal (GI) tract play an important role in maintaining host health and normal gut function [1-4]. The majority of studies of human intestinal microbiota have been performed under the assumption that faeces contain a representative sample of the prevalent intestinal microbiota. Traditionally, most of our knowledge regarding the activity of the GI microbiota has come from conventional culture based methods, requiring knowledge of bacterial growth conditions and several days for cultivation. This, together with the fact the only between 20-50% of intestinal bacteria can be cultured, led to the development of a more sensitive, specific and rapid molecular 16S DNA detection system based on the polymerase chain reaction (PCR) [1, 3-5]. However, the application of PCR to faecal samples has several pitfalls, as faeces contain numerous species of bacteria and several PCR inhibitors, most of which have not been identified [6, 7]. The difficulty with applying PCR methods to faeces has been attributed to the difficulty of removing inhibitors [8]. The extraction and purification of DNA from faecal samples largely influences the amount of these inhibitory substances. Phenol-chloroform extraction is the classical method for preparation of faecal samples for PCR assay, and inactivates microbes very efficiently [9]. However, phenol-chloroform extraction has been shown to be ineffective in removing all types of inhibitors from faecal samples, and residual phenol can inhibit amplification [9, 10]. Several commercially available faecal DNA extraction kits have been developed to overcome these limitations. These kits operate by different principles for the efficient lysis...
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