Simultaneous detection of Listeria spp. and Listeria monocytogenes by reverse hybridization with 16S-23S rRNA spacer probes

Abstract

Enzymatic amplification results showed that Listeria species have at least two 16S-23S rRNA spacer regions of different lengths. These spacer regions of L. monocytogenes, L. ivanovii and L. seeligeri were cloned after enzymatic amplification. Sequence analysis of the inserts revealed two spacers of 245-246 bp and 496-498 bp, respectively, of which the latter included tRNA(Ala) and tRNA(Ile) genes. One Listeria spp.-specific probe, LIS-ICG4, was deduced from the 245-bp spacer and a L. monocytogenes-specific probe, LMO-ICG5, was inferred from the 496-bp spacer. The specificity of both probes was tested in a reverse hybridization assay (Line Probe Assay, LiPA). Both LIS-ICG4 and LMO-ICG5 proved to be highly specific when hybridized to a large collection of Listeria strains and strains from other relevant taxa. The LiPA test herein described for the simultaneous detection of Listeria spp. and L. monocytogenes can be expanded to detect other foodborne pathogens.