title: Use of Whole-Genome Phylogeny and Comparisons for Development of a Multiplex PCR Assay To Identify Sequence Type 36 Vibrio parahaemolyticus.
abstract: Vibrio parahaemolyticus sequence type 36 (ST36) strains that are native to the Pacific Ocean have recently caused multistate outbreaks of gastroenteritis linked to shellfish harvested from the Atlantic Ocean. Whole-genome comparisons of 295 genomes of V. parahaemolyticus, including several traced to northeastern U.S. sources, were used to identify diagnostic loci, one putatively encoding an endonuclease (prp), and two others potentially conferring O-antigenic properties (cps and flp). The combination of all three loci was present in only one clade of closely related strains of ST36, ST59, and one additional unknown sequence type. However, each locus was also identified outside this clade, with prp and flp occurring in only two nonclade isolates and cps in four. Based on the distribution of these loci in sequenced genomes, prp identified clade strains with >99% accuracy, but the addition of one more locus increased accuracy to 100%. Oligonucleotide primers targeting prp and cps were combined in a multiplex PCR method that defines species using the tlh locus and determines the presence of both the tdh and trh hemolysin-encoding genes, which are also present in ST36. Application of the method in vitro to a collection of 94 clinical isolates collected over a 4-year period in three northeastern U.S. states and 87 environmental isolates revealed that the prp and cps amplicons were detected only in clinical isolates identified as belonging to the ST36 clade and in no environmental isolates from the region. The assay should improve detection and surveillance, thereby reducing infections.
authors.affiliation: Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, USA Northeast Center for Vibrio Disease and Ecology, University of New Hampshire, Durham, New Hampshire, USA cheryl.whistler@unh.edu.
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Whistler
authors.affiliation: Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, USA Northeast Center for Vibrio Disease and Ecology, University of New Hampshire, Durham, New Hampshire, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Hall
authors.affiliation: Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, USA Northeast Center for Vibrio Disease and Ecology, University of New Hampshire, Durham, New Hampshire, USA Genetics Graduate Program, University of New Hampshire, Durham, New Hampshire, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Xu
authors.affiliation: Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, USA.
authors.forename: Saba
authors.initials: S
authors.lastname: Ilyas
authors.affiliation: Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, USA.
authors.forename: Puskar
authors.initials: P
authors.lastname: Siwakoti
authors.affiliation: Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, USA Northeast Center for Vibrio Disease and Ecology, University of New Hampshire, Durham, New Hampshire, USA.
authors.forename: Vaughn S
authors.initials: VS
authors.lastname: Cooper
authors.affiliation: Northeast Center for Vibrio Disease and Ecology, University of New Hampshire, Durham, New Hampshire, USA Department of Natural Resources and the Environment, University of New Hampshire, Durham, New Hampshire, USA.
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Jones
mesh_terms: D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics*; D006801:Humans; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010802:Phylogeny; D014481:United States; D014735:Vibrio Infections / Q000175:diagnosis / Q000382:microbiology; D014736:Vibrio parahaemolyticus / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial
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