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

Abstract

Aims: To compare agar plate and real‐time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces. Methods and Results: 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−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® 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 QuantiTectTM SYBR® 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 × log(CFU) + 1·43 and log(DNA copy) = 1·62 × log(CFU) − 6·32 respectively. Conclusions: The real‐time PCR method could be used to enumerate Lactobacillus within 2 days when compared with plating method which requires 5–6 days. Significance and Impact of the Study: 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.

Effects of Short-Chain Fructooligosaccharides on Faecal Bifidobacteria and Specific Immune Response in Formula-Fed Term Infants: A Randomized, Double-Blind, Placebo-Controlled Trial

“…Faecal total bifidobacteria counts at M0, M2 and M3 were measured using quantitative Real-Time Polymerase Chain Reaction (RT PCR) (18)(19)(20)(21). Briefly, we used 1 mL aliquots of faecal samples to extract total DNA of bacterial populations by means of a commercial kit (QIAmp DNA Stool Mini Kit; Qiagene, Hilden, Germany).…”

Dominance of Lactobacillus acidophilus in the Facultative Jejunal Lactobacillus Microbiota of Fistulated Beagles

“…Due to sampling difficulty, the canine intestinal LAB microbiota has been studied mainly from feces (9,11). However, it is argued that fecal samples cannot represent the microbiota present in the upper gut since they are different niches and provide specific habitats for different microbiota (17).…”

Effect of Encapsulation on Viability of Pediococcus pentosaceus OZF During Its Passage Through the Gastrointestinal Tract Model

“…The significant correlation between the cfu of the OZF cells obtained by two methods indicated that it is possible to use DNA copy to estimate the number of bacteria. The plate counts and the results of qPCR assays were also found in good agreement for different target LAB species by Furet et al [12], Neeley et al [36], and Fu et al [11]. Additionally, Matsuda et al [32] determined the viable cell counts with a LIVE/DEAD BacLight bacterial viability kit, RT-qPCR, and conventional culture methods.…”