Purification of polymerase chain reaction (PCR)-amplifiable DNA from compost piles containing bovine mortalities.
Livestock production systems utilize composting as a method of disposal of livestock mortalities, but there is limited information on the rate and extent of carcass decomposition. Detection of specific DNA fragments by PCR offers a method for investigating the degradation of carcasses and other biological materials during composting. However, the purity of extracted DNA is critical for successful PCR analysis. We applied a method to purify DNA from compost samples and have tested the method by analyzing bovine and plant DNA targets after 0, 4, and 12 month of composting. The concentration of organic matter from composted material posed a particular challenge in obtaining pure DNA for molecular analysis. Initially extracted DNA from composted piles at day 147 was discoloured, and PCR inhibitors prevented amplification of target plant or bovine gene fragments. Bovine serum albumin improved detection by PCR (25-50 microl final volume) through the removal of inhibitors, but only when concentrations of humic acids in extracted DNA were 1.0 ng microl(-1) or less. Optimal purification of DNA from compost was achieved by chromatography using Sepharose 4B columns. The described DNA purification protocol enabled molecular monitoring of otherwise cryptic bovine and plant target genes throughout the composting process. The assay could likely be used to obtain PCR-amplifiable DNA that could be used for the detection of microbial pathogens in compost.
Reuter
Tim
T
Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, Canada.
Xu
Weiping
W
Alexander
Trevor W
TW
Stanford
Kim
K
Xu
Yongping
Y
McAllister
Tim A
TA
D000822:Animal Husbandry / Q000379:methods; D000818:Animals; D001709:Biotechnology / Q000379:methods; D002417:Cattle; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D004247:DNA / Q000032:analysis*; D006812:Humic Substances / Q000032:analysis; D010944:Plants / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012037:Refuse Disposal; D012710:Serum Albumin, Bovine / Q000032:analysis; D013997:Time Factors
D006812:Humic Substances; D012710:Serum Albumin, Bovine; D004247:DNA