A 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; and Biological Function Research Department, Food Science & Technology Institute, Morinaga Milk Industry Co., Ltd., 5-1-83, Higashihara, Zama, Kanagawa 228-8583, Japan

^* To whom correspondence should be addressed. Email: t_soezim{at}morinagamilk.co.jp.

	Abstract

Ethidium monoazide (EMA) is a DNA cross-linking agent and eukaorytic topoisomerase II poison. We previously reported that treatment of EMA with visible light irradiation (EMA+Light) directly cleaved chromosomal DNA of E. coli (Microbiol. Immunol. 51:763-775, 2007). Herein, we report that EMA+Light randomly cleaved chromosomal DNA of heat-treated, but not live, Listeria monocytogenes within 10 min of treatment. When polymerase chain reaction (PCR) amplified DNA of 894 bp, 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 within 30 min, to cleave chromosomal DNA by poisoning activity. PCR product of the hly gene from 10⁸ heat-treated L. monocytogenes was inhibited by a combination of 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. monocytognenes present in human blood. EMA+Light+T-poisons completely suppressed PCR product from 10³ - 10⁷ antibiotic-treated L. monocytogenes, 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 spiked into pasteurized milk. EMA+Light+T-poisons inhibited PCR product from 10³ – 10⁷ heat-treated cells, but could detect 10¹ live L. monocytogenes. Our method is useful in clinical as well as food-hygiene tests.