PCR–Restriction Fragment Length Polymorphism and Pulsed-Field Gel Electrophoresis Characterization of Listeria monocytogenes Isolates from Ready-to-Eat Foods, the Food Processing Environment, and Clinical Samples in South Africa

Abstract

ABSTRACT Listeria monocytogenes is a ubiquitous, intracellular foodborne pathogen that is responsible for invasive listeriosis. The ability of L. monocytogenes to cause disease has some correlation with the serotypes of a specific lineage group, making the identification of lineage groups important for epidemiological analysis. The development of typing methods to link the strains of L. monocytogenes to an outbreak of listeriosis would help minimize the spread of the disease. The aim of this study was to design a PCR–restriction fragment length polymorphism (RFLP) method to differentiate between the lineage groups of L. monocytogenes. PCR-amplified fragments of the hly gene for 12 serotypes of L. monocytogenes were sequenced, aligned, and analyzed with the BioEdit program, and single nucleotide polymorphisms (SNPs) within regions of this gene were identified. Because of the difficulty in acquiring a serotype 4ab reference strain, this serotype was not included in this study. We tested the specificity and accuracy of the PCR-RFLP method on these L. monocytogenes reference strains and validated the method with 172 L. monocytogenes strains recovered from humans, food, and the food processing environment in 2000 to 2002 and 2008 to 2010 from regions within South Africa. PCR-RFLP analysis applied in this study placed L. monocytogenes serotypes into one of three lineage groups based on the sequence differences and SNPs within each lineage group. The SNPs were conserved in a region where RFLP analysis could be applied for a distinction between L. monocytogenes lineage groups. HIGHLIGHTS