Development and application of a heterologous internal standard for polymerase-chain-reaction-based detection of Campylobacter jejuni and Campylobacter coli in foods

Abstract

 A heterologous internal standard, termed "mimic", was developed for the polymerase chain reaction (PCR)-based detection of Campylobacter jejuni and Campylobacter coli in food. Mimic was designed to contain a heterologous DNA fragment of plasmid pUC18, flanked by a primer binding site, identical to the bacterial target DNA. Application of mimic in the PCR permitted its co-amplification together with the bacterial DNA with similar efficiency. As the length of the amplified products differed, they were easily detectable by agarose gel electrophoresis. The presence or absence of the mimic PCR product was indicative of the efficacy of the PCR. The use of approximately 60 mimic molecules per reaction was optimal for determining the reliability of the diagnostic PCR assays without decreasing the detection limit. This system for the detection of the two species of Campylobacter was successfully applied in routine food surveillance.