Molecular detection methods for the investigation of potential sources of Campylobacter infection

Best, Emma Louisa (2004). Molecular detection methods for the investigation of potential sources of Campylobacter infection. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f9f0

Abstract

The rapid detection and identification of Campylobacter jejuni isolates to probable strain level would significantly inform the epidemiological investigation of C.jejuni infection. At the outset of this project the molecular fingerprinting techniques PFGE and AFLP were proven to be equally discriminatory for identification of outbreak strains of Campylobacter, however both techniques were time consuming and not directly applicable to potential sources of infection. Real time PCR approaches were pursued for the purpose of developing methods, which would be specific and robust for the detection of specific strains of Campylobacter. A duplex real-time polymerase chain reaction (PCR) assay for speciation of Campylobacter jejuni and C.coli using real time platforms was developed. This enabled a turnaround time of three hours and was applied for direct spéciation from sources of infection including meat samples. The development of real time PCR assays for allelic discrimination of strain associated single nucleotide polymorphisms (SNPs) based upon MLST locus alleles offered a possible approach for rapid strain detection. Single nucleotide polymorphisms defining key alleles diagnostic for six major clonal complexes were identified, following a detailed analysis of the available MLST data. Allelic discrimination assays based on real time PCR systems were designed to detect the SNPs and be specific for clonal complexes ST-21, ST-45, ST-48, ST-61 ST-206 and ST-257. SNP based assays were evaluated using panels of isolates from human infections, poultry, the environment, and the MLST reference collection, which had previously been characterized by MLST. Real time allelic discrimination assays allowed the rapid detection of C.jejuni isolates and preliminary strain identification directly from foods and environmental specimens. The ability to combine detection with the identification of epidemiologically important information beyond genus or species identification represents a major new concept in the use of nucleic acid amplification techniques for the improved detection of pathogens particularly pathogens of major public health importance such as C.jejuni.

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