Multiplex riboprobes for the detection of virulent Yersinia enterocolitica and simple methods for their preparation.
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.
Fliss
I
I
Département de Sciences et Technologie des Aliments, Université Laval, Québec, Canada.
Blais
B W
BW
Holley
R
R
Simard
R E
RE
D018829:Adhesins, Bacterial / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D004268:DNA-Binding Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004755:Enterobacteriaceae / Q000235:genetics; D004768:Enterotoxins / Q000235:genetics; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015347:RNA Probes / Q000235:genetics*; D014764:Viral Proteins; D037521:Virulence Factors; D015008:Yersinia enterocolitica / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity
D018829:Adhesins, Bacterial; C069712:Ail protein, Yersinia enterocolitica; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004768:Enterotoxins; D015347:RNA Probes; D014764:Viral Proteins; D037521:Virulence Factors; C064832:YadA protein, Yersinia; C048669:heat-stable enterotoxin (Yersinia); C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases