title: Rapid detection of viable Escherichia coli O157 by coupling propidium monoazide with loop-mediated isothermal amplification.
abstract: Conventional molecular detection methods cannot distinguish between viable and dead Escherichia coli O157 cells. In this study, the loop-mediated isothermal amplification (LAMP) method combined with propidium monoazide (PMA) treatment was developed to selectively detect viable E. coli O157 cells. Four primers, including outer primers and inner primers, were specially designed for the recognition of six distinct sequences on the serogroups (O157) of the specific rfbE gene of the E. coli O157 genome. PMA selectively penetrated through the compromised cell membranes and intercalated into DNA. Amplification of DNA from dead cells was completely inhibited by 3.0 μg/ml PMA, whereas the DNA derived from viable cells was amplified remarkably within 1 h by PMA-LAMP. Exhibiting high sensitivity and specificity, PMA-LAMP is a suitable method for evaluating the inactivation efficacy of slightly acidic electrolyzed water in broth. PMA-LAMP can selectively detect viable E. coli O157 cells. This study offers a novel molecular detection method to distinguish between viable and dead E. coli O157 cells.
authors.affiliation: Department of Food Science and Biotechnology and Institute of Bioscience and Biotechnology, Kangwon National University.
authors.forename: Xihong
authors.initials: X
authors.lastname: Zhao
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Fereidoun
authors.initials: F
authors.lastname: Forghani
authors.forename: Joong-Hyun
authors.initials: JH
authors.lastname: Park
authors.forename: Myoung-Su
authors.initials: MS
authors.lastname: Park
authors.forename: Kun-Ho
authors.initials: KH
authors.lastname: Seo
authors.forename: Deog-Hwan
authors.initials: DH
authors.lastname: Oh
mesh_terms: D001386:Azides / Q000378:metabolism*; D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D019453:Escherichia coli O157 / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D050296:Microbial Viability; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives* / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D001386:Azides; D017931:DNA Primers; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; C533957:propidium monoazide; D011419:Propidium