論文 - 詳細
| RRC ID | 15647 |
|---|---|
| 著者 | Soejima T, Iida K, Qin T, Taniai H, Seki M, Yoshida S. |
| タイトル | Method To Detect Only Live Bacteria during PCR Amplification. |
| ジャーナル | J Clin Microbiol |
| 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. |
| 巻・号 | 46(7) |
| ページ | 2305-13 |
| 公開日 | 2008-7-1 |
| DOI | 10.1128/JCM.02171-07 |
| PII | JCM.02171-07 |
| PMID | 18448692 |
| PMC | PMC2446937 |
| MeSH | Azides / metabolism Bacterial Toxins / genetics Colony Count, Microbial / methods* DNA Topoisomerase IV / antagonists & inhibitors DNA, Bacterial / metabolism Enzyme Inhibitors / pharmacology Heat-Shock Proteins / genetics Hemolysin Proteins / genetics Hot Temperature Humans Light Listeria monocytogenes / genetics* Microbial Viability* Polymerase Chain Reaction / methods* RNA, Ribosomal, 23S / genetics Sensitivity and Specificity Topoisomerase II Inhibitors |
| IF | 5.897 |
| 引用数 | 63 |
| WOS 分野 | MICROBIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 11 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 一般微生物 | JCM 2873 |