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Journal of Clinical Microbiology, June 2001, p. 2227-2232, Vol. 39, No. 6
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.6.2227-2232.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

J. M. J. Logan, K. J. Edwards, N. A. Saunders, and J. Stanley*

Molecular Biology Unit, Virus Reference Division, Central Public Health Laboratory, London, NW9 5HT, United Kingdom

Received 23 October 2000/Returned for modification 23 January 2001/Accepted 8 April 2001

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.

* Corresponding author. Mailing address: Molecular Biology Unit, Virus Reference Division, Central Public Health Laboratory, 61 Colindale Ave., London, NW9 5HT, United Kingdom. Phone: 44 20 82004400. Fax: 44 20 82001569. E-mail: jlogan{at}hgmp.mrc.ac.uk.

Journal of Clinical Microbiology, June 2001, p. 2227-2232, Vol. 39, No. 6
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.6.2227-2232.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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