Comparison of agar plate and real-time PCR on enumeration of Lactobacillus, Clostridium perfringens and total anaerobic bacteria in dog faeces

To compare agar plate and real-time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces. Thirty-two faecal specimens from Labrador retriever dogs were used to compare agar plate and real-time PCR enumeration methods for Lactobacillus, C. perfringens and total anaerobic bacteria. Total anaerobic bacteria, C. perfringens and Lactobacillus of faeces were counted (as CFU g[superscript [-]1] faeces) for 48-h incubation at 37°C in an anaerobic gas chamber on genus-selective media. Total genomic DNA from samples was extracted by the QIAamp[superscript ®] DNA stool mini kit. The quantification of DNA (as DNA copy per gram faeces) by real-time PCR was performed with a LightCycler system with the QuantiTect[superscript TM] SYBR[superscript ®] green PCR kit for PCR amplification. The results indicated that there was a significant correlation between CFU and DNA copy of Lactobacillus (R² = 0·78, P < 0·01) and total anaerobic bacteria (R² = 0·21, P < 0·05); but no correlation was found between CFU and DNA copy of C. perfringens. The regression equations for Lactobacillus and total anaerobic bacteria were log(DNA copy) = 0·83 [multiplication] log(CFU) + 1·43 and log(DNA copy) = 1·62 [multiplication] log(CFU) [-] 6·32 respectively. The real-time PCR method could be used to enumerate Lactobacillus within 2 days when compared with plating method which requires 5-6 days. The real-time PCR method and the primer set for Lactobacillus spp. harboured in the dog intestine can be used for rapid enumeration of lactobacilli and monitoring of the faecal Lactobacillus community.