title: Method To Detect Only Live Bacteria during PCR Amplification.
abstract: Ethidium monoazide (EMA) is a DNA cross-linking agent and eukaryotic topoisomerase II poison. We previously reported that the treatment of EMA with visible light irradiation (EMA + Light) directly cleaved chromosomal DNA of Escherichia coli (T. Soejima, K. Iida, T. Qin, H. Taniai, M. Seki, A. Takade, and S. Yoshida, Microbiol. Immunol. 51:763-775, 2007). Herein, we report that EMA + Light randomly cleaved chromosomal DNA of heat-treated, but not live, Listeria monocytogenes cells within 10 min of treatment. When PCR amplified DNA that was 894 bp in size, PCR final products from 10(8) heat-treated L. monocytogenes were completely suppressed by EMA + Light. When target DNA was short (113 bp), like the hly gene of L. monocytogenes, DNA amplification was not completely suppressed by EMA + Light only. Thus, we used DNA gyrase/topoisomerase IV and mammalian topoisomerase poisons (here abbreviated as T-poisons) together with EMA + Light. T-poisons could penetrate heat-treated, but not live, L. monocytogenes cells within 30 min to cleave chromosomal DNA by poisoning activity. The PCR product of the hly gene from 10(8) heat-treated L. monocytogenes cells was inhibited by a combination of EMA + Light and T-poisons (EMA + Light + T-poisons), but those from live bacteria were not suppressed. As a model for clinical application to bacteremia, we tried to discriminate live and antibiotic-treated L. monocytogenes cells present in human blood. EMA + Light + T-poisons completely suppressed the PCR product from 10(3) to 10(7) antibiotic-treated L. monocytogenes cells but could detect 10(2) live bacteria. Considering the prevention and control of food poisoning, this method was applied to discriminate live and heat-treated L. monocytogenes cells spiked into pasteurized milk. EMA + Light + T-poisons inhibited the PCR product from 10(3) to 10(7) heat-treated cells but could detect 10(1) live L. monocytogenes cells. Our method is useful in clinical as well as food hygiene tests.
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. t_soezim@morinagamilk.co.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Soejima
authors.forename: Ken-ichiro
authors.initials: K
authors.lastname: Iida
authors.forename: Tian
authors.initials: T
authors.lastname: Qin
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Taniai
authors.forename: Masanori
authors.initials: M
authors.lastname: Seki
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Yoshida
mesh_terms: D001386:Azides / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics; D015169:Colony Count, Microbial / Q000379:methods*; D027101:DNA Topoisomerase IV / Q000037:antagonists & inhibitors; D004269:DNA, Bacterial / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D008027:Light; D008089:Listeria monocytogenes / Q000235:genetics*; D050296:Microbial Viability; D016133:Polymerase Chain Reaction / Q000379:methods*; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012680:Sensitivity and Specificity; D059005:Topoisomerase II Inhibitors
chemical_list: D001386:Azides; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D012338:RNA, Ribosomal, 23S; D059005:Topoisomerase II Inhibitors; C014535:8-azidoethidium; D027101:DNA Topoisomerase IV; C038705:hlyA protein, Listeria monocytogenes