Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR
Article first published online: 18 MAY 2014
© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Volume 3, Issue 4, pages 437–445, August 2014
How to Cite
MicrobiologyOpen 2014; 3(4): 437–445
- Issue published online: 14 AUG 2014
- Article first published online: 18 MAY 2014
- Manuscript Accepted: 7 APR 2014
- Manuscript Revised: 1 APR 2014
- Manuscript Received: 8 JAN 2014
- French Agency for Food, Environmental and Occupational Health and Safety
- French Environment and Energy Management Agency
- Irstea-Région Bretagne Fellowship
- DNA extraction kit;
- elution procedure;
- Lactobacillus amylovorus ;
- pig manure;
- total bacteria
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.