Development of species-specific DNA probes for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari by polymerase chain reaction fingerprinting

Abstract

The application of polymerase chain reaction (PCR) fingerprinting assays enables discrimination between species and strains of microorganisms. PCR primers aiming at arbitrary sequences in combination with primers directed against the repetitive extragenic palindrome (REP) or enterobacterial repetitive intergenic consensus (ERIC) motifs generate isolate-specific DNA banding patterns. Analysis of these PCR fingerprints obtained for 33 isolates of Campylobacter jejuni, 30 isolates of Campylobacter coli, and 8 isolates of Campylobacter lari revealed that besides generation of isolate-specific fragments, species-specific DNA fragments of identical size were synthesized. It appeared that these DNA fragments could be used as species-specific probes, since they are unique for the pattern which they are deriving from. The probes do not cross-react with amplified DNA originating from a large panel of nonrelated microorganisms. Moreover, these probes displayed species specificity, as they reacted with a single restriction fragment on Southern blots containing DNA from C. jejuni, C. coli, and C. lari and other Campylobacter species. This combination of PCR fingerprinting and probe hybridization results in a highly specific identification assay and provides an example of specific test development without the prior need for DNA sequence information. The principle of the procedure holds great promise for the rapid isolation of DNA probes which, in combination with a general PCR assay, may lead to efficient typing and detection procedures for a multitude of medically important nonviral microorganisms.

Rapid identification of diverse Campylobacter lari strains isolated from mussels and oysters using a reverse hybridization line probe assay

“…Several DNA-based methods have been developed for the detection and identification of Campylobacter species in food, clinical and environmental samples (Oyofo et al 1992;Oyofo and Rollins 1993;Eyers et al 1993;Uyttendaele et al 1994;Korolik et al 1995;Stucki et al 1995;Cardarelli-Leite et al 1996;Jackson et al 1996;Giesendorf et al 1992Giesendorf et al , 1993. Although these methods are faster than conventional methods, the efficacy is often limited, since they require multiple PCR reactions per sample or digestions with multiple restriction enzymes.…”

Campylobacter jejuni: Specific oligonucleotides and DNA probes for use in polymerase chain reaction-based diagnosis

“…Other workers have already developed PCR tests for the detection of C. jejuni, but C. coli was also detected [15,16]. In a recent study [17], the PCR fingerprinting technique was successfully applied to the specific detection of C. jejuni. However, this PCR fingerprinting method used alone may not be sufficiently reliable for diagnostic purposes.…”

“…Nevertheless, none of these methods can be used for the specific identification of C. jejuni, since cross-reactions with 338 other thermophilic Campylobacter species were observed. More recently, a PCR-fingerprinting method was described for the development of specific DNA probes for C. jejuni, C. coli and C. lari [17]. However, the sequences of these probes were not determined, and species-specificity seems to depend upon the stringency conditions used.…”

Microbes in food processing technology

“…This rRNA gene restriction pattern (ribotype) approach is increasingly used to build molecular identification or typing schemes. More recently, rapid PCR-typing methods evolved that are based on random amplification of polymorphic DNA (RAPD) [48,49], on amplification of restriction fragment polymorphisms [50,51], or on amplification with primers derived from conserved repetitive sequences: REP-PCR (REP stands for Repetitive Extragenic Palindromic element) and ERIC-PCR (ERIC stands for Enterobacterial Repetitive Intergenic Consensus sequence) [52,53] or eukaryotic repeated sequence motives [54,55].…”