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

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.

Electronic deoxyribonucleic acid (DNA) microarray detection of viable pathogenic Escherichia coli, Vibrio cholerae, and Salmonella typhi.

An electronic deoxyribonucleic acid (DNA) microarray technique was developed for detection and identification of viable Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella typhi. Four unique genes, the E. coli O157 lipopolysaccharide (LPS) gene (rfbE) and H7 flagellin gene (fliC), the V. cholerae O1 LPS gene (rfbE), and the S. typhi LPS gene (tyv), were chosen as the targets for detection. These targets were selectively amplified from mRNA of viable cells using reverse transcription polymerase chain reaction (RT-PCR) and detected using the electronic DNA microarray technique. Specific captures and reporters were designed and examined for selective detection and correct identification of the target pathogens. The technique was able to detect as few as 2-150 cells of E. coli O157:H7. The co-presence of six other common bacteria and a parasite at 10- and 1000-fold higher concentrations than the target E. coli O157:H7 did not interfere with the specific detection. Comparative analysis of live and heat-killed E. coli O157:H7 cells showed that the technique only responded to the viable cells and not to the dead cells. Thus, the integration of RT-PCR of specific mRNA with the electronic DNA microarray technique enables specific and sensitive detection of viable target cells. This technique is potentially useful for high throughput screening of multiple pathogenic bacteria in different samples.