Identification of lactic acid bacteria isolated from poultry carcasses by high-resolution melting (HRM) analysis

Abstract

Lactic acid bacteria (LAB) are a complex group of Gram-positive bacteria belonging to different genera with common morphological, metabolic and physiological characteristics. Their classification was initially based on biochemical methods, but nowadays molecular methods are usually applied for the identification of LAB. Herein, real-time PCR assay coupled with high-resolution melting (HRM) analysis was developed for identifying and distinguishing LAB isolates coming from poultry carcasses. The 16S rRNA gene from these isolates was amplified using primers that annealed to conserved regions. The melting curve analysis of the amplicons classified all isolates into ten LAB species and generated ten distinct HRM curve profiles. The results from HRM analysis were compared to those produced by API 50 CH biochemical microkits and ribosomal DNA sequencing, suggesting the superiority of HRM against API. In conclusion, HRM was proven to be a fast, reliable and cost-effective method for identification of LAB isolates. HRM analysis could be used in order to reduce the time needed for the identification assay and the cost of sequencing the entire group of LAB isolates. The melting profile of known LAB species could be used as a reference for the rapid identification of unknown LAB isolates without the need of sequencing.