High resolution melting analysis for the characterization of lineage II Listeria monocytogenes serovars 1/2a and 1/2c based on single nucleotide polymorphisms identification within the Listeria Pathogenicity Island-1 and inlAB operon: a novel approach for epidemiological surveillance.

Abstract

A high resolution melting (HRM) assay was developed for characterizing lineage II Listeria monocytogenes based on the amplification and the melting profiles analysis of 81 fragments targeting the region from the prs to ldh loci, including the Listeria Pathogenicity Island-1 (LIPI-1) genes and the inlAB operon. Real-time PCR and HRM protocols were standardized using 10 replicate assays from L. monocytogenes EGD-e reference strain (serovar 1/2a). Twenty wild-type isolates of serovar 1/2a and two of serovar 1/2c were tested, and differences between EGD-e strain and the wild-type isolates were defined if the melting temperature (Tm ) of an amplicon was not within the lower and the upper limits calculated from replicate testing on EGD-e. The analysis revealed 17 and 19 HRM profiles with respect to prs/LIPI-1/ldh and inlAB target regions (Simpson's Index of Diversity 0·979 and 0·983) respectively. The 1/2c cultures showed 98·1% similarity to melting characteristics with EGD-e, whilst 1/2a isolates had the greatest heterogeneity that was related to inlA, inlB and actA genes. Sequencing of amplicons generating different Tm values from EGD-e confirmed the presence of point mutations. This method was useful for L. monocytogenes subtyping based on single nucleotide polymorphisms detection through the melting behaviour analysis of main virulence genes. The study underlines the effectiveness of HRM in differentiating L. monocytogenes strains with high discriminatory power, thus rendering it useful for epidemiological surveillance.