A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

摘要

cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate.