title: 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.
abstract: Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type.
authors.affiliation: Chapman University, School of Earth and Environmental Sciences, Food Science and Nutrition, Orange, CA 92866, USA. hellberg@chapman.edu
authors.forename: Rosalee S
authors.initials: RS
authors.lastname: Hellberg
authors.forename: Keely G
authors.initials: KG
authors.lastname: Martin
authors.forename: Ashley L
authors.initials: AL
authors.lastname: Keys
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Haney
authors.forename: Yuelian
authors.initials: Y
authors.lastname: Shen
authors.forename: R Derike
authors.initials: RD
authors.lastname: Smiley
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D002611:Cheese / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics*; D005506:Food Contamination / Q000032:analysis; D005638:Fruit / Q000382:microbiology; D008087:Listeria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017747:Seafood / Q000382:microbiology; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA gene; DNA sequencing; Listeria; MicroSeq ID; Molecular subtyping