title: A rapid typing method for Listeria monocytogenes based on high-throughput multilocus sequence typing (Hi-MLST).
abstract: Listeria monocytogenes infects humans via food products, causing listeriosis. Consequently, food companies pay meticulous attention to the risk of contamination of their products by this bacterium. While fragment analysis methods such as pulsed-field gel electrophoresis (PFGE) are used to trace the sources of contamination for this bacterium, some drawbacks have been identified, namely the complexity of the methods and the difficulty of making data comparisons. As an alternative, multilocus sequence typing (MLST) is now seeing widespread use; however, owing to its cost, time, and labor requirements, its diffusion into the food industry has been slow. Thus, in the present study, a High-throughput MLST (Hi-MLST) method, which can rapidly, simply, and cheaply perform MLST analyses using a next-generation sequencer (NGS) that can analyze a large volume of base sequences at once was developed. Firstly, a multiplex PCR method designed to amplify seven genes for use in MLST was developed. The discriminatory potential of the developed method was confirmed in silico, and was verified that it has the same discriminatory potential as conventional methods. Next, MLST analysis using multiplex PCR and NGS was performed for 48 strains of L. monocytogenes. The sequences obtained from this analysis have sufficiently reliable quality for all of the genes from of all the strains. Thus, this method could classify the 48 strains into 39 sequence types (ST) with a Diversity index (DI) of 0.989. In summary, using the Hi-MLST method developed in the present study, which combined multiplex PCR and NGS, cut the costs to 1/6th and the time to 1/20th that of conventional MLST methods.
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan. Electronic address: hajime@kaiyodai.ac.jp.
authors.forename: Hajime
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Iwakawa
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Ohshima
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kyoui
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
authors.forename: Shiori
authors.initials: S
authors.lastname: Kumano
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kuda
authors.affiliation: Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
authors.forename: Bon
authors.initials: B
authors.lastname: Kimura
mesh_terms: D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D020043:Genes, Essential / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008089:Listeria monocytogenes / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D008088:Listeriosis / Q000175:diagnosis / Q000382:microbiology; D058885:Multilocus Sequence Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods
keywords: Listeria monocytogenes; Molecular typing; Multilocus sequence typing; Next generation sequencing