Physical Pre-Treatment Improves Efficient DNA Extraction and qPCR Sensitivity from Clostridium Difficile Spores in Faecal Swine Specimens.

Abstract

A considerable fraction of the faecal microbiota is spore-forming. Molecular quantification of bacteria may be underestimated if preceded with nucleic acid extraction without special treatment to extract recalcitrant bacterial spores. The objective of this study was to improve the DNA extraction regarding the presence of Clostridium difficile spores in faecal swine specimens. Sow faeces were inoculated with spores of C. difficile (10(6)CFU), frozen at -30°C overnight and subjected to DNA extraction. As a preceding step to a standard DNA extraction method (QIAamp DNA stool Mini kit), different physical treatments such as microwave oven heating and repeated bead-beating techniques and a combination of both were applied and compared with each other by means of qPCR. Using a standard DNA extraction method only, C. difficile spores were quantified at 4.96 log copy number/200mg of faeces. A repeated bead-beating at 6m/s for 10min followed by a standard DNA extraction resulted in 5.77 log copy number of spores in inoculated faeces. Heating in a microwave oven at 800W for 1, 3, 5 and 10min followed by a standard DNA extraction resulted in a gene quantification of up to 4.89 log copy number. A combination of both methods resulted in the bacterial gene quantity of 5.37 log copy number. Pre-treatment with repeated bead-beating led to the highest quantification of bacteria, and therefore it can be applied for more efficient DNA extraction from spores of C. difficile in faecal specimens.