title: Triplex PCR for the simultaneous detection of Pseudomonas aeruginosa, Helicobacter hepaticus, and Salmonella typhimurium.
abstract: The accurate and economical diagnosis of pathogenic bacteria is necessary for the microbiological control of laboratory animals. In this study, we developed a triplex PCR method for the direct detection of three common gastroenteric bacteria, Pseudomonas aeruginosa, Helicobacter hepaticus, and Salmonella typhimurium. Targets were specifically amplified by conventional PCR assay using a genomic fragment from P. aeruginosa, 16S ribosomal RNA from H. hepaticus, and the invA gene from S. typhimurium. To investigate the specificity of our primers, they were tested against purified DNA from many other bacterial species. There were no amplification products from other bacteria. Under optimized conditions, the triplex assay simultaneously yielded a 726-bp product from P. aeruginosa, a 417-bp product from H. hepaticus, and a 246-bp product from S. typhimurium. The detection limits of this assay in pure culture were 10 pg for P. aeruginosa, and 0.1 pg for H. hepaticus and S. typhimurium. All three bacteria were successfully detected in the liver, cecum, and feces of experimentally infected mice. This method is a useful and convenient assay that allows the simultaneous identification of bacterial pathogens in mice. Our triplex method will be used to improve quality control in the detection of pathogenic bacterial infections in laboratory animal facilities.
authors.affiliation: Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Japan.
authors.forename: Eui-Suk
authors.initials: ES
authors.lastname: Jeong
authors.forename: Kyoung-Sun
authors.initials: KS
authors.lastname: Lee
authors.forename: Seung-Ho
authors.initials: SH
authors.lastname: Heo
authors.forename: Jin-Hee
authors.initials: JH
authors.lastname: Seo
authors.forename: Yang-Kyu
authors.initials: YK
authors.lastname: Choi
mesh_terms: D000818:Animals; D000830:Animals, Laboratory; D001424:Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology*; D001431:Bacteriological Techniques / Q000379:methods*; D044923:Helicobacter hepaticus / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000302:isolation & purification*; D012486:Salmonella typhimurium / Q000302:isolation & purification*; D012680:Sensitivity and Specificity