GENOMIC SUBTRACTION IN COMBINATION WITH PCR FOR ENRICHMENT OF LISTERIA MONOCYTOGENES-SPECIFIC SEQUENCES

Genomic DNA from Listeria innocua and Listeria ivanovii was used in su btractive hybridization with DNA from Listeria monocytogenes involving two amplification strategies. Subtraction was accomplished by labelli ng the subtracting DNA with biotin and removal after liquid hybridizat ion with tester DNA (L. monocytogenes) by reaction with streptavidin a nd 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-s pecific primer; in both amplifications, the amplified products were la belled with biotin-modified dUTP. Southern hybridization of the amplif ied/subtracted probes with tester- and subtracter-related strains demo nstrated numerous L. monocytogenes-specific sequences. The genome-subt racted mixed probe identified 7 RFLP patterns among 13 strains of L. m onocytogenes representing 11 L. monocytogenes serovars. Southern blot analysis demonstrated that the subtracted probe cross-hybridized to tw o bands among L. welshimeri strains but had little or no hybridization with five other species of Listeria including L. innocua, L. ivanovii , 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 enri ched sequences in the subtracted probe may be useful for typing L. mon ocytogenes strains by specific RFLP patterns or for cloning L. monocyt ogenes-specific sequences.