Evaluation and comparison of DNA extraction kits for the detection of clostridium difficile in spiked and field faeces from piglets by using real-time PCR

Abstract

In complex samples (faeces, soil, food, etc.), Clostridium difficile is often present in the form of dormant spores that cause reduced effectiveness of DNA extraction. With the aim of determining an optimal DNA extraction procedure from spores, DNA extraction from faecal samples spiked with a known number of C. difficile spores and faecal samples from piglets was performed with three manual protocols, using two commercial kits and subsequent real-time PCR (rtPCR) DNA amplification. DNA extraction protocols, including mechanical disruption by bead beating, gave better results with rtPCR. The SmartHelix DNAid Complex Kit proved to be more efficient than the QIAamp DNA Stool Mini Kit, suggesting that an optimal combination of mechanical, enzymatic, and chemical lysis seems to be required for the best results.