Konventionelle PCR- und Real-Time PCR- Verfahren zum Nachweis von thermophilen Campylobacter jejuni, C. coli und C. lari : ein Überblick

Abstract

Many PCR-techniques have already been published for the detection of thermophilic Campylobacters. In order to provide a practical overview for the user – without laying claim to completeness – we present a collection of actual PCR detection methods, some of them were modified and some are still unpublished. They are listed along with their target genes and specificities. Four conventional and seven real-time PCR methods were tested and compared using a panel of 65 Campylobacter strains belonging to 12 different more or less related species. The regular PCR methodes tested include the 16S rRNA gene based technique that specifically detects C. jejuni, C. coli and C. lari without differenciating to species level, two flaA gene based typing-methods, one for C. jejuni and C. coli in combination, which was slightly modified according to Nachamkin et al. (1993, 1996), and additionally for C. lari. Another duplex PCR was carried out which allows identification of the two species C. jejuni and C. coli simultaneously. Among the real-time methods tested there were three techniques allowing identification of C. jejuni alone, two assays based on the flagellin A gene capable to identify C. jejuni and C. coli in combination, a duplex PCR performed as triplex PCR combined with an internal amplification control that detected C. jejuni and C. coli separately, and a Lightcycler method that detected C. jejuni, C. coli and C. lari simultaneously. Inclusivity and exclusivity were shown for each of the methods. In most cases inclusivity was good, but some techniques raised problems concerning exclusivity. Flagellin based real-time PCR techniques partially crossreacted either with C. lari or C. upsaliensis. C. hyoilei which is very closely related to C. coli reacted in almost all methods applied like C. coli.