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A rapid detection system specific for Listeria monocytogenes based upon the polymerase chain reaction was developed. The specificity of the primers and the probe annealing to the coding region of the mpl gene proved positive with the DNA from a total of 103 L. monocytogenes strains, while DNA from another 73 Listeria and non-Listeria strains tested negative. To facilitate detection with large numbers of samples, a microtitre plate assay was established with biotinylated probes. Use of a standard DNA prevented false-negative results when used as an internal amplification control in the PCR-ELISA. As the described method required approximately 5-6 h to be completed it may prove useful in the detection of L. monocytogenes in food.

Yersinia enterocolitica is an important zoonosis, which can cause disease in humans and animals. The culture methods available for detection of Y. enterocolitica in food samples are time-consuming and seldom successful. Using DNA-based methods, like PCR, this pathogen can be detected more rapidly and with greater sensitivity. The aim of this study was to establish a rapid and accurate real-time PCR method to detect pathogenic Y. enterocolitica in pork. The chromosomal ail-gene, which is only present in pathogenic Y. enterocolitica strains, was used as DNA target for the 5' nuclease PCR assay. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). A 2-step protocol with 45 cycles was used in a multicolour real-time PCR detection system. A Ct value over 40 indicated a negative result. The DNA extraction procedure for the natural samples was rapid and simply. This qualitative real-time PCR method was shown to be specific and sensitive. Detection rate of ail-positive Y. enterocolitica in 200 pig tonsils was 88 % and 35 % with PCR and culture methods, respectively. When 100 raw pork samples were studied, 7 were positive with PCR and all were culture negative.Zusammenfassung (Redaktion): Yersinia enterocolitica ist als Pathogen von Bedeutung für Mensch und Tier. Die Kultur-Methoden, die für den Nachweis von Y. enterocolitica in Lebensmittel-Proben zur Verfügung stehen, sind Zeit-aufwendig und selten erfolgreich. Wenn DNA-basierte Methoden wie zum Beispiel die PCR angewandt werden, kann dieser pathogene Mikroorganismus schneller und besser nachgewiesen werden. In diesem Bericht wird eine schnelle und sehr spezifische real-time PCR vorgestellt, um Y. enterocolitica in Schweinefleisch nachzuweisen. Das chromosomale ail-Gen, das nur in pathogener Y. enterocolitica vorkommt, wurde als DNA-Sonde für den 5`Nuklease-PCR-Assay eingesetzt. Die Probe wurde am 5`-Ende mit dem fluoreszierenden FAM und am 3`-Ende mit dem Quencher TAMRA markiert. Ein Verfahren -bestehend aus zwei Abschnitten -mit 45 Zyklen wurde in einem Multicoulor real-time-PCR NachweisSystem durchgeführt. Ct-Werte größer als 40 stehen für negative Ergebnisse. Die DNA-Extraktion aus den Proben war schnell und einfach. Die hier beschriebene real-time PCR ist spezifisch und sensitiv. Die Nachweisrate für ail-positive Y. enterocolitica in 200 Schweinemandeln lag bei 88 % bei der PCR-Methode und bei 35 % bei Kulturmethoden. Bei 100 Proben rohem Schweinefleisch waren mit Hilfe der PCR 7 Proben positiv, aber mit Hilfe der Kulturmethoden alle negativ.

A specific PCR-ELISA detection system was established for the testing of food products for Salmonella contamination. Initially a shortened microbial enrichment comprising two steps, a non-selective and selective incubation each of 8 hours at 37 °C, is carried out. DNA is then isolated from 1 ml of the final selective enrichment culture and purified. In vitro amplification of the recovered DNA is performed in a competitive PCR in the presence of internal standard DNA to exclude the possibility of false negative results arising from food-borne inhibitory factors. Salmonella specific amplicons were identified through specific probe Correspondence and reprint requests to:

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