Detection of pathogenic Yersinia enterocolitica in foods and water by immunomagnetic separation, nested polymerase chain reactions, and colorimetric detection of amplified DNA

Abstract

A two-step polymerase chain reaction (PCR) procedure with two nested pairs of primers specific for theyad4 gene of Yersinia enterocolitica was developed. The PCR assay identified all common pathogenic serogroups

Detection of Yersinia enterocolitica O:3 in faecal samples and tonsil swabs from pigs using IMS and PCR

“…A more rapid enrichment may be obtained at a higher temperature, using selective media (Wauters et al 1988). Growth at higher temperature has previously been used to enrich Y. enterocolitica before detection with IMS-PCR (Kapperud et al 1993). Using a nonselective medium and overnight incubation at room temperature, the assay could detect samples spiked with 2 cfu g-' food, but only when the background flora was below lo7 cfu m1-I.…”

“…Moreover, IMS removes inhibiting material both in the removal of the supernatant fluid and due to the washing of the immunomagnetic particles. The combination of IMS and PCR has previously been used to concentrate and detect Listeria monocytogenes from three naturally contaminated cheese samples (Fluit et al 1993a), Salmonella from spiked samples of poultry (Fluit et al 1993b) and from 14 naturally infected human faecal samples (Widjojoatmodjo et al 1992), Y. enterocolitica from spiked food and water samples (Kapperud et al 1993), Escherichia coli from five naturally infected and several spiked pig stool specimens and Chlamydia trachomatis from 27 naturally infected urine samples .…”

New methods of diagnosis in plant pathology – perspectives and pitfalls 1

“…bacteria, viruses) are first trapped from a solution by antibodies coated on magnetic beads and the beads trapped by a magnet. Subsequently PCR can be performed on the concentrated antigens (Wetzel et al, 1992;Kapperud et al, 1993). Another example of immuno-PCR is a method where antibodies directed to RNA-DNA hybrids may be used for sensitive detection of PCR products.…”

“…In practice, however, sensitivity is usually lower. To enhance specificity, 'nested' PCR may be performed: a first primer pair is used to multiply a larger fragment of the target, then a second primer pair is used to recognize and multiply a smaller part of the amplified sequence (White et al, 1992;Yourno, 1992;Henson & French, 1993;Kapperud et al, 1993). To verify identy of PCR products, hybridization with probes via blotting is often applied.…”

“…PCR is also performed in combination with immunomagnetic capture (Kapperud et a/., 1993;Grant eta/., 1993) and fluorescent labels (Cano et al, 1993) in order to increase sensitivity and/or specificity. In some tests, an ELISA-plate set-up is achieved (White et a/., 1992;Galindo et al, 1993).…”