Rapid Identification of Campylobacter spp. by Melting Peak Analysis of Biprobes in Real-Time PCR

Abstract

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 basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of Campylobacter, Helicobacter, and Arcobacter 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 taxa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from 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 microaerobic culture.

Molecular Characterization of Diarrheagenic Bacteria Isolated from Stool of Under-five Children in Dar Es Salaam. Tanzania

“…The results, which reveal a significant proportion of PCR-positive but culture-negative specimens were consistent with many other studies (Logan, et al 2001, Gentry-Weeks, et al 2002, Kulkarni, et al 2002. Taken together with the epidemiological data presented in the present study, supported the view that PCR-based testing is more sensitive than conventional culture for the detection of enteric bacterial pathogens in stool samples.…”

Campylobacter

“…C. concisus, C. curvus, C. gracilis, C. rectus and C. showae are detected in association with the oral cavity [82]. Alternatively, C. mucosalis, C. helveticus and C. sputorum biovar faecalis are isolated from animals [114,161].…”

“…hyointestinalis, C. fetus and C. mucosalis [81]. Rapid PCR-biprobe identification scheme based on the realtime PCR was developed for the Campylobacter taxa pathogenic for humans, including C. lanienae [82]. This new organism has also been isolated from the faeces of six healthy pigs in Japan [148] and from the faeces of bovine and beef cattle, in the beef cattle, C. lanienae was the most frequently detected species (49%), in Canada [62,63,115,118].…”

Gene expression analysis in calcific tendinopathy of the rotator cuff

“…Quantifi cation of tissue transglutaminase 2 (tTG2), collagen X, collagen V, VEGF, Matrix Metalloprotease 9 (MMP9), osteopontin, osteonectin, catepsin K was performed using the Sybr green method (Logan et al, 2009). Primers were designed spanning an intron-exon junction, and their sequences are reported in Table 1. For each PCR reaction, 15 ng of cDNA were used as template.…”