Most Probable Number Method Combined with Nested Polymerase Chain Reaction for Detection and Enumeration of Enterotoxigenic Clostridium perfringens in Intestinal Contents of Cattle, Pig and Chicken

Abstract

The most probable number (MPN) method combined with a nested polymerase chain reaction (nested PCR) for the detection and enumeration of enterotoxigenic Clostridium perfringens in the intestinal contents of cattle, pig and chicken was examined. Ten-fold serial dilutions of samples were added to three tubes of enrichment medium, which were incubated at 37°C for 20-24 hr, and the C. perfringens enterotoxin gene was detected by nested PCR from the enrichment culture without isolating the organism. The results obtained by this method with artificially contaminated intestinal contents were significantly correlated with those obtained by a plate count method. When the method was applied 10 the detection and enumeration of indigenous enterotoxigenic C. perfringens, the organism was found in two, two and three samples of 10 intestinal contents of cattle, pig and chicken, respectively. Most of the positive samples contained fewer than 10 MPN/g of enterotoxigenic C. perfringens, except one sample of chicken, which contained 1.5 × 102 MPN/g. The MPN method combined with nested PCR is easy to perform and may be a useful tool for the detection and enumeration of enterotoxigenic C. perfringens in intestinal contents.