title: Melting curve analysis of a groEL PCR fragment for the rapid genotyping of strains belonging to the Lactobacillus casei group of species.
abstract: Lactobacillus casei group (Lcs) consists of three phylogenetically closely related species (L. casei, L. paracasei, and L. rhamnosus), which are widely used in the dairy and probiotic industrial sectors. Strategies to easily and rapidly characterize Lcs are therefore of interest. To this aim, we developed a method according to a technique known as high resolution melting analysis (HRMa), which was applied to a 150 bp groEL gene fragment. The analysis was performed on 53 Lcs strains and 29 strains representatives of species that are commonly present in dairy and probiotic products and can be most probably co-isolated with Lcs strains. DNA amplification was obtained only from Lcs strains, demonstrating the specificity of the groEL primers designed in this study. The HRMa clustered Lcs strains in three groups that exactly corresponded to the species of the L. casei group. A following HRMa separated the 39 L. paracasei strains in two well distinct intraspecific groups, indicating the possible existence of at least two distinct genotypes inside the species. Nonetheless, the phenotypic characterization demonstrated that the genotypes do not correspond to the two L. paracasei subspecies, namely paracasei and tolerans. In conclusion, the melting curve analysis developed in this study is demonstrably a simple, labor-saving, and rapid strategy obtain the genotyping of a bacterial isolate and simultaneously potentially confirm its affiliation to the L. casei group of species. The application of this method to a larger collection of strains may validate the possibility to use the proposed HRMa protocol for the taxonomic discrimination of L. casei group of species. In general, this study suggests that HRMa can be a suitable technique for the genetic typization of Lactobacillus strains.
authors: Nepal Academy of Science and Technology (NAST), Khumaltar, Lalitpur, Nepal; Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal.
authors: Ranjan
authors: R
authors: Koirala
authors: Department of Food, Environmental and Nutritional Sciences (DeFENS), Division of Food Microbiology and Bioprocessing, Università degli Studi di Milano, Italy.
authors: Valentina
authors: V
authors: Taverniti
authors: Department of Food, Environmental and Nutritional Sciences (DeFENS), Division of Food Microbiology and Bioprocessing, Università degli Studi di Milano, Italy.
authors: Silvia
authors: S
authors: Balzaretti
authors: Department of Food, Environmental and Nutritional Sciences (DeFENS), Division of Food Microbiology and Bioprocessing, Università degli Studi di Milano, Italy.
authors: Giovanni
authors: G
authors: Ricci
authors: Department of Food, Environmental and Nutritional Sciences (DeFENS), Division of Food Microbiology and Bioprocessing, Università degli Studi di Milano, Italy.
authors: Maria Grazia
authors: MG
authors: Fortina
authors: Department of Food, Environmental and Nutritional Sciences (DeFENS), Division of Food Microbiology and Bioprocessing, Università degli Studi di Milano, Italy. Electronic address: simone.guglielmetti@unimi.it.
authors: Simone
authors: S
authors: Guglielmetti
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics; D002611:Cheese / Q000382:microbiology; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D005243:Feces / Q000382:microbiology; D005838:Genotype; D006801:Humans; D007422:Intestines / Q000382:microbiology; D007780:Lacticaseibacillus casei / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009055:Mouth / Q000382:microbiology; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D044366:Transition Temperature
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D017931:DNA Primers; D004269:DNA, Bacterial
keywords: HRM analysis; Lactobacillus; Molecular typing; Probiotic