APPLICATION OF A COLONY PCR TECHNIQUE WITH FUNG'S DOUBLE TUBE METHOD FOR RAPID DETECTION AND CONFIRMATION OFCLOSTRIDIUM PERFRINGENS

Abstract

ABSTRACTA colony polymerase chain reaction (PCR) technique was applied with an established Fung's double tube (FDT) method for rapid detection and confirmation ofClostridium perfringens.Published sequences of PCR primers forC. perfringensalpha toxin gene were used and PCR conditions were optimized. From the detection ofC. perfringensby FDT tube to the confirmation by a colony PCR assay took as short as 16–18 h. The method was applied to 147 isolates of anaerobic sulfite reducing bacteria isolated from foods, sewages and animal clinical specimens. The results were compared with standard methods for the confirmation ofC. perfringens.Of those 147 suspected isolates, 97 and 99 were confirmed asC. perfringensby standard methods and the colony PCR technique, respectively. We found the developed method simple, rapid, cost‐effective, and most importantly, very reliable for the detection and confirmation ofC. perfringens.PRACTICAL APPLICATIONSWith Fung's double tube method and a colony polymerase chain reaction technique, a completed determination ofClostridium perfringenscontamination can be accomplished within 16–18 h. This saves at least 2–3 days when compared with standard methods. Moreover, it minimizes the cost and labor needed since an anaerobic chamber as well as steps of Gram staining and biochemical testing can be avoided. The developed method is a powerful tool and an alternative for the enumeration ofC. perfringens. It can be applied to samples from various sources and highly reliable results are expected. Most microbiological laboratories have a thermocycler and reagents as parts of their basic instruments. Therefore, the developed method can be easily applied without massive investment.