Multiplex riboprobes for the detection of virulent Yersinia enterocolitica and simple methods for their preparation

J Appl Bacteriol. 1995 Aug;79(2):195-202. doi: 10.1111/j.1365-2672.1995.tb00935.x.

Abstract

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 plasma-borne sequences permitted detection of virulent Y. enterocolitica, regardless of plasmid loss during handling of cultures, and was unreactive with a virulent 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.

MeSH terms

  • Adhesins, Bacterial / genetics
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Proteins / genetics*
  • Bacterial Toxins / genetics
  • Bacterial Typing Techniques
  • Base Sequence
  • DNA, Bacterial / analysis*
  • DNA-Binding Proteins / genetics
  • DNA-Directed RNA Polymerases / chemistry
  • Enterobacteriaceae / genetics
  • Enterotoxins / genetics
  • Immunoblotting
  • Molecular Sequence Data
  • Nucleic Acid Hybridization
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • RNA Probes / genetics*
  • Viral Proteins
  • Virulence Factors*
  • Yersinia enterocolitica / classification
  • Yersinia enterocolitica / genetics
  • Yersinia enterocolitica / isolation & purification*
  • Yersinia enterocolitica / pathogenicity

Substances

  • Adhesins, Bacterial
  • Ail protein, Yersinia enterocolitica
  • Bacterial Outer Membrane Proteins
  • Bacterial Proteins
  • Bacterial Toxins
  • DNA, Bacterial
  • DNA-Binding Proteins
  • Enterotoxins
  • RNA Probes
  • Viral Proteins
  • Virulence Factors
  • YadA protein, Yersinia
  • heat-stable enterotoxin (Yersinia)
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases