This Article Full Text Full Text (PDF) Other Versions of this Article: JCM.02171-07v1 46/7/2305    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Google Scholar Articles by Soejima, T. Articles by Yoshida, S.-i. PubMed PubMed Citation Articles by Soejima, T. Articles by Yoshida, S.-i.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, July 2008, p. 2305-2313, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.02171-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Method To Detect Only Live Bacteria during PCR Amplification

Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan,¹ Biological Function Research Department, Food Science & Technology Institute, Morinaga Milk Industry Co., Ltd., 5-1-83, Higashihara, Zama, Kanagawa 228-8583, Japan²

Received 9 November 2007/ Returned for modification 23 December 2007/ Accepted 19 April 2008

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 10⁸ 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 10⁸ 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 10³ to 10⁷ antibiotic-treated L. monocytogenes cells but could detect 10² 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 10³ to 10⁷ heat-treated cells but could detect 10¹ live L. monocytogenes cells. Our method is useful in clinical as well as food hygiene tests.

Published ahead of print on 30 April 2008.