Rapid identification of Listeria species and screening for variants by melting curve and high-resolution melting curve analyses of the intergenic spacer region of the rRNA gene

Abstract

The presence of any Listeria species in food may be an indicator of poor hygiene in food processing facilities. The biochemical identification of Listeria species is laborious and time consuming. Therefore, the development of novel identification methods that are rapid and simple to perform would be an asset. In this study, large intergenic spacer region amplicons of 343-374 bp were generated from 207 Listeria isolates. The melting curve analysis of these amplicons specifically classified all isolates into 6 Listeria species and generated 11 high-resolution melting (HRM) curve profiles. In this study, 3 HRM profiles were found in Listeria monocytogenes and Listeria innocua, and 2 were found in Listeria seeligeri. Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence. The smallest difference recognized in this study was 1 nt. The results represented in this study show that HRM curve analysis of Listeria intergenic spacer sequences is a simple, quick, and reproducible method of simultaneously identifying 6 Listeria species and screening for variants. In particular, the completion of both reaction and analysis in a closed tube saves time by eliminating the separate steps and lowers the risk of contamination.