Listeria monocytogenes lineage group classification by MAMA-PCR of the listeriolysin gene

Nucleotide sequence differences within several virulence genes, including t he listeriolysin O (hly) gene, are associated with three evolutionary linea ge groups of Listeria monocytogenes. Because the ability of L. monocytogene s to cause disease may vary by evolutionary lineage group, rapid discrimina tion among the three lineage types may be important for estimating pathogen ic potential. A Mismatch Amplification Mutation Assay (MAMA) was developed and used to rapidly screen and characterize L. monocytogenes isolates with regard to lineage type. A standard PCR amplified a 446-bp region within the hly gene with all three L. monocytogenes lineage genotypes. MAMA primers t o four different sites within this region of the hly gene were designed to amplify under the same PCR conditions and generated amplicons, the size of which depended on the isolate genotype. Ninety-seven L. monocytogenes isola tes were screened. All isolates, except ATCC 19116, could be classified by MAMA PCR as one of the three hly genotypes. Overall, 56, 36, and 4 of the 9 7 isolates tested were type 1, 2, or 3 respectively. Among the 26 patient i solates, 85%, 15%, and 0% were type 1, 2, or 3 respectively; for the 60 foo d isolates, 54% were type 1, 43% were type 2, and 3% were type 3. The combi nation of these MAMA PCR analyses provides a rapid method to screen and cat egorize L. monocytogenes isolates because of conserved nucleotide differenc es within the hly gene.