An electronic DNA microarray technique for detection and differentiation of viable Campylobacter species

Abstract

An electronic oligonucleotide microarray technique was developed for detection and differentiation of the viable Campylobacter species, C. jejuni, C. coli, and C. lari. This development consisted of four major components: identification of single nucleotide polymorphisms (SNPs) within the hsp60 gene as species markers, design of fluorescently labelled SNP-based reporters, development of an electronic microarray detection, and application of the integrated technique to analysis of Campylobacter species in food samples. A unique capability of this technique is the specific detection of viable cells and not dead ones. This is achieved by using mRNA of the 60 kDa heat-shock protein as the viability marker. The identification of two unique SNPs closely located at positions 291 and 294 of the hsp60 gene enabled the differentiation of the three Campylobacter species. This technique was able to detect as few as two viable Campylobacter cells. The analysis of 19 blind Campylobacter samples showed 100% agreement with their identities obtained using pulsed-field gel electrophoresis. The analysis of six chicken samples revealed the presence of C. coli in one of the samples.

Comparative Analysis of New Zealand Campylobacter Isolates Using MLST, PFGE and flaA PCR RFLP Genotyping

“…Based on DNA hybridisation studies, sequencing and protein analysis, biochemical characteristics and growth conditions, the Campylobacter genus is recognised as containing 18 different species and six sub-species (76,106,131,168,218,238). Classification of the species within the Campylobacter genus by the "gold standard" (204) of DNA-DNA hybridisation has been augmented by AFLP fingerprinting (57), MLEE using 11 housekeeping enzymes (144), microarray analysis using SNPs in the hsp60 gene (238), MLST using seven housekeeping genes (136) and sequencing of the 16S rRNA (76,106), 23S rRNA (106), cpn60 (106) and rpoB genes (125). Several studies have noted that due to high levels of rRNA sequence conservation between bacterial species, classification using sequencing of the 16S rRNA gene alone may lead to misclassification of isolates (85,112).…”

Comparative Analysis of New Zealand Campylobacter Isolates Using MLST, PFGE and flaA PCR RFLP Genotyping

“…Based on DNA hybridisation studies, sequencing and protein analysis, biochemical characteristics and growth conditions, the Campylobacter genus is recognised as containing 18 different species and six sub-species (76,106,131,168,218,238). Classification of the species within the Campylobacter genus by the "gold standard" (204) of DNA-DNA hybridisation has been augmented by AFLP fingerprinting (57), MLEE using 11 housekeeping enzymes (144), microarray analysis using SNPs in the hsp60 gene (238), MLST using seven housekeeping genes (136) and sequencing of the 16S rRNA (76,106), 23S rRNA (106), cpn60 (106) and rpoB genes (125). Several studies have noted that due to high levels of rRNA sequence conservation between bacterial species, classification using sequencing of the 16S rRNA gene alone may lead to misclassification of isolates (85,112).…”