Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR

We describe rapid PCR-biprobe identification of Campylobacter spp.. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the bas is of their degree of hybridization to three 16S ribosomal DNA (rDNA) bipro bes. First-round symmetric PCR is performed with genus-specific primers whi ch selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a Li ghtCycler in the presence of one of three biprobes; the identity of an ampl ified DNA-biprobe duplex is established after determination of the species- specific melting peak temperature. The biprobe specificities were determine d by testing 37 reference strains of Campylobacter, Helicobacter, and Arcob acter spp. and 59 Penner serotype reference strains of Campylobacter jejuni and C. coli. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five ta xa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hy ointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code wa s broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly f rom fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR-enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following m icroaerobic culture.