A real-time multiplexed PCR assay for rapid detection and differentiation of Campylobacter jejuni and Campylobacter...

Campylobacter species are the leading agents of bacterial gastroenteritis worldwide. C. jejuni and C. coli together are responsible for more than 95% of all cases of Campylobacter-induced diarrheal disease in the United States. Detection of campylobacteria in clinical samples by conventional culture is problematic and slow due to their complex taxonomy, fastidious growth requirements, and biochemical inertness. The current study describes a rapid, sensitive, and specific real-time polymerase chain reaction (PCR) assay capable of detecting and differentiating C. jejuni (hippuricase gene, hipO) and C. coli (serine hydroxymethyltransferase gene, glyA) in a single reaction, directly from clinical isolates and human feces. The analytical specificity of the assay was demonstrated with a diverse range of Campylobacter species, related organisms, and other diarrhea-inducing bacterial pathogens. The analytical sensitivity of the multiplexed, PCR assay was 10 genome copies for both C. jejuni and C. coli. Following a rapid DNA extraction method (QIAGEN QIAamp DNA stool Mini Kit), the multiplexed PCR identified C. jejuni or C. coli in 100% of fecal samples containing 10(3) colony-forming units (CFU) per gram of feces. This assay represents the first real-time PCR method capable of detecting and differentiating C. jejuni and C. coli in a single reaction.