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


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 degrees 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 QuantiTect 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 x log(CFU) + 1.43 and log(DNA copy) = 1.62 x 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.


CONCLUSIONS
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.
Fu
C J
CJ
Department of Animal Sciences, University of Missouri, Columbia, MO 65211, USA. cfad0@mizzou.edu
Carter
J N
JN
Li
Y
Y
Porter
J H
JH
Kerley
M S
MS
D000818:Animals; D001421:Bacteria, Anaerobic / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D003016:Clostridium perfringens / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D015169:Colony Count, Microbial / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004285:Dogs / Q000382:microbiology; D005145:Face / Q000382:microbiology*; D007778:Lactobacillus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D013223:Statistics as Topic
D004269:DNA, Bacterial