title: Development of a rapid SNP-typing assay to differentiate Bifidobacterium animalis ssp. lactis strains used in probiotic-supplemented dairy products.
abstract: Identification at the genus, species, and strain levels is desirable when a probiotic microorganism is added to foods. Strains of Bifidobacterium animalis ssp. lactis (BAL) are commonly used worldwide in dairy products supplemented with probiotic strains. However, strain discrimination is difficult because of the high degree of genome identity (99.975%) between different genomes of this subspecies. Typing of monomorphic species can be carried out efficiently by targeting informative single nucleotide polymorphisms (SNP). Findings from a previous study analyzing both reference and commercial strains of BAL identified SNP that could be used to discriminate common strains into 8 groups. This paper describes development of a minisequencing assay based on the primer extension reaction (PER) targeting multiple SNP that can allow strain differentiation of BAL. Based on previous data, 6 informative SNP were selected for further testing, and a multiplex preliminary PCR was optimized to amplify the DNA regions containing the selected SNP. Extension primers (EP) annealing immediately adjacent to the selected SNP were developed and tested in simplex and multiplex PER to evaluate their performance. Twenty-five strains belonging to 9 distinct genomic clusters of B. animalis ssp. lactis were selected and analyzed using the developed minisequencing assay, simultaneously targeting the 6 selected SNP. Fragment analysis was subsequently carried out in duplicate and demonstrated that the assay yielded 8 specific profiles separating the most commonly used commercial strains. This novel multiplex PER approach provides a simple, rapid, flexible SNP-based subtyping method for proper characterization and identification of commercial probiotic strains of BAL from fermented dairy products. To assess the usefulness of this method, DNA was extracted from yogurt manufactured with and without the addition of B. animalis ssp. lactis BB-12. Extracted DNA was then subjected to the minisequencing protocol, resulting in a SNP profile matching the profile for the strain BB-12.
authors.affiliation: Dipartimento di Scienze Veterinarie, Università degli Studi di Torino, 10095 Grugliasco TO, Italy; Department of Food Science, The Pennsylvania State University, University Park 16802.
authors.forename: Sara
authors.initials: S
authors.lastname: Lomonaco
authors.affiliation: Department of Food Science, The Pennsylvania State University, University Park 16802.
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Furumoto
authors.affiliation: Department of Food Science, The Pennsylvania State University, University Park 16802.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Loquasto
authors.affiliation: Dipartimento di Scienze Veterinarie, Università degli Studi di Torino, 10095 Grugliasco TO, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Morra
authors.affiliation: Dipartimento di Scienze Veterinarie, Università degli Studi di Torino, 10095 Grugliasco TO, Italy.
authors.forename: Ausilia
authors.initials: A
authors.lastname: Grassi
authors.affiliation: Department of Food Science, The Pennsylvania State University, University Park 16802. Electronic address: rfr3@psu.edu.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Roberts
mesh_terms: D000818:Animals; D001483:Base Sequence; D001644:Bifidobacterium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D003611:Dairy Products / Q000382:microbiology; D005260:Female; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D019936:Probiotics / Q000032:analysis*; D017422:Sequence Analysis, DNA; D015014:Yogurt / Q000382:microbiology*
keywords: Bifidobacterium animalis ssp. lactis; molecular subtyping; probiotic; single nucleotide polymorphism (SNP) minisequencing