title: An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses.
abstract: Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10(-2)-5.6 × 10(-5) (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.
authors.affiliation: Shimane Prefectural Institute of Public Health and Environmental Science.
authors.forename: Jun
authors.initials: J
authors.lastname: Kawase
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Etoh
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ikeda
authors.forename: Keiji
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Masanori
authors.initials: M
authors.lastname: Watahiki
authors.forename: Tomoko
authors.initials: T
authors.lastname: Shima
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Kameyama
authors.forename: Kazumi
authors.initials: K
authors.lastname: Horikawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fukushima
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Goto
authors.forename: Komei
authors.initials: K
authors.lastname: Shirabe
mesh_terms: D015373:Bacterial Typing Techniques; D052160:Benzothiazoles; D016123:Campylobacter jejuni / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics*; D003959:Diamines; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology; D005456:Fluorescent Dyes; D005517:Foodborne Diseases / Q000175:diagnosis* / Q000382:microbiology; D057166:High-Throughput Screening Assays; D006801:Humans; D057230:Limit of Detection; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009930:Organic Chemicals; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000302:isolation & purification
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I