title: Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples.
abstract: Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.
authors.affiliation: Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, USA.
authors.forename: Si Hong
authors.initials: SH
authors.lastname: Park
authors.forename: Irene
authors.initials: I
authors.lastname: Hanning
authors.forename: Robin
authors.initials: R
authors.lastname: Jarquin
authors.forename: Philip
authors.initials: P
authors.lastname: Moore
authors.forename: Dan J
authors.initials: DJ
authors.lastname: Donoghue
authors.forename: Ann M
authors.initials: AM
authors.lastname: Donoghue
authors.forename: Steven C
authors.initials: SC
authors.lastname: Ricke
mesh_terms: D058491:Bacterial Load / Q000379:methods*; D052160:Benzothiazoles; D016123:Campylobacter jejuni / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012486:Salmonella typhimurium / Q000302:isolation & purification*; D013194:Staining and Labeling / Q000379:methods; D044366:Transition Temperature; D014481:United States; D014871:Water Microbiology
chemical_list: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I