A SIMPLE RNA PROBE SYSTEM FOR ANALYSIS OF LISTERIA-MONOCYTOGENES POLYMERASE CHAIN-REACTION PRODUCTS

The synthesis of an RNA probe specific for the hlyA gene of Listeria m onocytogenes by in vitro transcription from a polymerase chain reactio n (PCR)-generated template incorporating bacteriophage T7 promoter seq uences is described. This simple method produced a high yield of RNA w hich hybridized specifically with hlyA PCR products on a membrane, res ulting in RNA-DNA hybrids which were detected by an immunoenzymatic as say with an anti-RNA-DNA hybrid antibody. The RNA probe hybridization system was more sensitive in the analysis of the PCR products than was the conventional agarose gel electrophoresis method. When applied to the analysis of PCR samples from cultures of various Listeria and non- Listeria organisms, the RNA probe was reactive in the assay of 62 diff erent L. monocytogenes isolates but not other Listeria species. Among the non-Listeria organisms tested, only Enterococcus faecalis gave a w eak positive reaction with more than 10(9) cells per ml. This reactivi ty disappeared at lower cell densities. This strategy for the synthesi s and application of RNA probes should facilitate the analysis of PCR products in the detection of L. monocytogenes and possibly other food pathogens.