MULTIPLEX RIBOPROBES FOR THE DETECTION OF VIRULENT YERSINIA ENTEROCOLITICA AND SIMPLE METHODS FOR THEIR PREPARATION

摘要

A Simple multiplex riboprobe system for detection of virulent Yersinia enterocolitica was developed using a pool of RNA probes specific for various chromosomal and plasmid-borne virulence gene sequences (yadA, virC, ail and yst). Riboprobes were synthesized by a rapid, simple and efficient technique involving in vitro transcription of polymerase chain reaction-generated templates incorporating bacteriophage T7 RNA polymerase promoter sequences in one of the priming oligonucleotides. After dot blotting target DNA samples on nitrocellulose and hybridization with the riboprobes, the RNA : DNA hybrids formed were detected by a simple immunoenzymic assay involving sequential reactions with an anti-RNA : DNA hybrid monoclonal antibody, anti-mouse Ig-peroxidase conjugate and chromogenic or chemiluminescent enzyme substrate solution. This multiplex riboprobe system targeting both chromosomal and plasmid-borne sequences permitted detection of virulent Y. enterocolitica, regardless of plasmid loss during handling of cultures, and was unreactive with avirulent Y. enterocolitica, other Yersinia and other bacteria. This system resulted in a significant improvement in the limit of detection in comparison to that obtained with individual probes.