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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 {triangledown}

Takashi Soejima,1,2* Ken-ichiro Iida,1 Tian Qin,1 Hiroaki Taniai,1 Masanori Seki,1 and Shin-ichi Yoshida1

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.

* Corresponding author. Mailing address: Biological Function Research Department, Food Science & Technology Institute, Morinaga Milk Industry Co., Ltd., 5-1-83, Higashihara, Zama, Kanagawa 228-8583, Japan. Phone: 81-46-252-3047. Fax: 81-46-252-3055. E-mail: t_soezim{at}morinagamilk.co.jp

{triangledown} Published ahead of print on 30 April 2008.

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.



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