Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real‐time PCR

Jérémy Desneux,Anne‐Marie Pourcher

doi:10.1002/mbo3.178

Abstract

Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil®, PowerFecal®, NucleoSpin® Soil kits and QIAamp® DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin® Soil kit (NS kit), and to a lesser extent the PowerFecal® kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure.

Highlights

Pig manure hosts a complex community of microorganisms that may vary during storage and during aerobic or anaerobic digestion
Cultivation methods are usually used to evaluate the impact of manure treatment on pathogens and on indicator bacteria whereas molecular methods are more appropriate to identify the dominant microbial groups and shifts in the community composition (Leung and Topp 2001; Peu et al 2006; Spence et al 2008; Liu et al 2009; Barret et al 2012) and to detect or quantify specific markers of pig manure contamination (Ufnar et al 2007; Marti et al 2009, 2010; Mieszkin et al 2009; Arthurson 2011)
The aims of this study were to compare the ability of four commercial DNA extraction kits to quantify total bacteria and, a specific pig manure marker, Lactobacillus amylovorus

Summary

Introduction

Pig manure hosts a complex community of microorganisms that may vary during storage and during aerobic or anaerobic digestion. Two microbial aspects of manure treatment are the most frequently studied: (1) the health risks associated with pathogenic bacteria and (2) the role of the microorganisms in the transformation of the organic matter. Cultivation methods are usually used to evaluate the impact of manure treatment on pathogens and on indicator bacteria (fecal coliforms, enterococci) whereas molecular methods are more appropriate to identify the dominant microbial groups and shifts in the community composition (Leung and Topp 2001; Peu et al 2006; Spence et al 2008; Liu et al 2009; Barret et al 2012) and to detect or quantify specific markers of pig manure contamination (Ufnar et al 2007; Marti et al 2009, 2010; Mieszkin et al 2009; Arthurson 2011).

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Conclusion