Genomic subtraction in combination with PCR for enrichment of Listeria monocytogenes-specific sequences

Genomic DNA from Listeria innocua and Listeria ivanovii was used in subtractive hybridization with DNA from Listeria monocytogenes involving two amplification strategies. Subtraction was accomplished by labelling the subtracting DNA with biotin and removal after liquid hybridization with tester DNA (L. monocytogenes) by reaction with streptavidin and phenol extraction. In one strategy, L. monocytogenes DNA was poly(A)-tailed with terminal transferase and amplified asymmetrically after subtraction. In another procedure, adapters ligated to the target DNA allowed symmetrical amplification after subtraction using an adapter-specific primer; in both amplifications, the amplified products were labelled with biotin-modified dUTP. Southern hybridization of the amplified/subtracted probes with tester- and subtracter-related strains demonstrated numerous L. monocytogenes-specific sequences. The genome-subtracted mixed probe identified 7 RFLP patterns among 13 strains of L. monocytogenes representing 11 L. monocytogenes serovars. Southern blot analysis demonstrated that the subtracted probe cross-hybridized to two bands among L. welshimeri strains but had little or no hybridization with five other species of Listeria including L. innocua, L. ivanouii, L. seeligeri, L. grayi, and L. murrayi. These data demonstrate that genomic subtraction via subtractive hybridization is a powerful method to enrich for specie-specific sequences in L. monocytogenes; the enriched sequences in the subtracted probe may be useful for typing L. monocytogenes strains by specific RFLP patterns or for cloning L. monocytogenes-specific sequences.