Genotyping of Campylobacter jejuni using a novel Polymerase-Chain-Reaction-Microsphere approach

Abstract

Campylobacter is a major cause of foodborne disease, with Campylobacter jejuni contributing more than 90% of reported cases. For diagnosis and monitoring of transmission, several genotyping methods have been developed, including multi-locus sequence typing (MLST) [1]. However, there is still a need for fast, cost effective methods for routine analysis. We developed a technique combining allele-specific PCR with a microsphere-flow cytometer system [2]. Seven loci with single-nucleotide polymorphisms (SNPs) with the highest Simpson's index of diversity (D) were selected from an MLST database. With these loci as a target, multiplex allele-specific PCR was conducted on microspheres in a single reaction and fluorescence signal was detected in a flow cytometer. The signal on the microspheres indicated which allele specific primer was consumed in the PCR. By this means all seven loci could be determined. This approach has a turnaround time of 4 h.Results To date the method has been tested with two strains of Campylobacter jejuni possessing known SNP patterns and six of seven loci have been correctly determined for these strains in a single PCR reaction.Conclusion A new allele specific PCR-microsphere-based method for genotyping Campylobacter jejuni is under development. This approach will be useful in laboratories utilizing PCR and flow cytometers.