Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR.
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.
Irstea-Rennes, Rennes, France; Université Européenne de Bretagne, Rennes, France.
Jérémy
J
Desneux
Anne-Marie
AM
Pourcher
D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D001431:Bacteriological Techniques / Q000379:methods*; D058448:Biota; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D008372:Manure / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013552:Swine
D004269:DNA, Bacterial; D008372:Manure
DNA extraction kit; Lactobacillus amylovorus; elution procedure; pig manure; qPCR; total bacteria