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.

FIG. 1.

Gel electrophoresis patterns of chromosomal DNA purified from live and heat-treated L. monocytogenes before and after EMA + Light treatment. N, no EMA + Light treatment; E, EMA + Light treatment (at 4°C in the dark for 5 min; irradiation for 5 min); M1, λ-EcoT14 I digest; and M2, 100-bp DNA ladder. The experiments for DNA extraction were performed in duplicate, and the electrophoresis patterns were the same.

FIG. 2.

Amplification results of 23S rRNA gene PCR after no treatment or EMA + Light treatment using live and heat-treated L. monocytogenes. The amplification results for the 23S rRNA gene (894 bp) of L. monocytogenes are represented. N, no EMA + Light treatment; E, EMA + Light treatment (at 4°C in the dark for 5 min; irradiation for 5 min); M1, λ-EcoT14 I digest; and M2, 100-bp DNA ladder. The PCR experiments were done in three replicates, and the results were the same.

FIG. 3.

Amplification results of PCR that targeted the hly gene after the combination treatment of EMA + Light and CIN/mammalian topoisomerase (I and II) poisons on live and heat-treated L. monocytogenes. The targeted hly gene was 113 bp. T-poison treatments were carried out for 30 min after EMA + Light treatment. N, no EMA + Light treatment; E, EMA + Light treatment (at 4°C in the dark for 5 min; irradiation for 5 min); M, 100-bp DNA ladder; CIN, 4 μg/ml CIN; CAM, 10 μg/ml CAM; ETP, 10 μg/ml ETP; ELP, 0.5 μg/ml ELP; MIT, 1 μg/ml MIT; and m-AMSA, 10 μg/ml m-AMSA. The concentrations represent final concentrations. The experiments were carried out in triplicate, and the electrophoresis images were the same.

FIG. 4.

Effects of CIN on the retaining activities of DNA gyrase and topoisomerase IV in heat-treated L. monocytogenes. The influence of CIN on the chromosomal DNA of live and heat-treated L. monocytogenes and the effect of DNase retained in heat-treated bacteria on the chromosomal DNA of heat-treated L. monocytogenes is shown. N, no treatment; CIN (+), CIN treatment. CIN (−) represents that heat-treated L. monocytogenes cells were incubated at 30°C for 24, 48, or 72 h without CIN. The evaluations were done in duplicate, and the same electrophoresis patterns were obtained.

FIG. 5.

Detection limit of L. monocytogenes and the discrimination of the live or antibiotic-treated L. monocytogenes cells in human blood by PCR that targeted the hly gene. (A) Live L. monocytogenes (1.8 × 10⁰ to 1.8 × 10⁷ cells/ml) inoculated into heparinized healthy human blood was harvested, and the hly (listeriolysin O) gene (113 bp; short DNA) was targeted by PCR. (B to D) Live or antibiotic-treated L. monocytogenes cells were mixed in human blood, and then treatments with EMA + Light + T-poisons and PCR methods were carried out. M, 100-bp DNA ladder; hly, listeriolysin O gene (113 bp; short DNA) of L. monocytogenes; N, no treatment, as a control; E, EMA + Light (at 4°C in the dark for 5 min; irradiation for 5 min); CAM, 25 μg/ml CAM; ETP, 25 μg/ml ETP; ELP, 2.5 μg/ml ELP; and m-AMSA, 25 μg/ml m-AMSA. The concentrations represent final concentrations. Each examination was performed in duplicate, and the same results were observed.

FIG. 6.

Detection limit of L. monocytogenes and discrimination of live or heat-treated cells spiked into pasteurized milk with PCR that targeted the hly gene. (A) Live L. monocytogenes spiked into pasteurized (125°C for 2 s) commercial milk (2.2 × 10⁰ to 2.2 × 10⁷ cells/ml) was recovered as bacterial pellets and supplied to a direct PCR (hly) cocktail. The hly (listeriolysin O) gene (113 bp; short DNA) was targeted by PCR. (B and C) Live or heat-treated L. monocytogenes was spiked in milk and then treated by EMA + Light + T-poisons and PCR methods. M, 100-bp DNA ladder; hly, listeriolysin O gene (113 bp; short DNA) of L. monocytogenes; N, no treatment, as a control; E, EMA + Light (at 4°C in the dark for 5 min; irradiation for 5 min); CAM, 25 μg/ml CAM; ETP, 25 μg/ml ETP; ELP, 2.5 μg/ml ELP; and m-AMSA, 25 μg/ml m-AMSA. The concentrations represent final concentrations. The experiments were carried out in duplicate, and the results were reproducible. UHT, ultra-high-temperature pasteurized.

FIG. 7.

(Left) Scheme for PCR suppression by EMA, psoralen, and a methylisopsoralen derivative (4′-AMDMIP) as a DNA cross-linking agent (current scheme). (Right) Scheme for PCR suppression through DNA cleavage by a new function of EMA and T-poisons containing fluoroquinolone (new scheme). Dark red bar, EMA, psoralen, or 4′-AMDMIP; yellow bar, T-poison (m-AMSA, etc.); Topo IV, bacterial topoisomerase IV. The cleavage points are represented in double-stranded DNA.