An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates.

Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.