| YANG Qianqian,ZHANG Xuzhi,JIANG Xiaoyu,LI Yang,ZHAO Jun,HAO Zhihui,WANG Pingping,QU Keming.Direct Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay.渔业科学进展,2020,41(2):41-50 |
| Direct Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay |
| Direct Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay |
| 投稿时间：2019-02-20 修订日期：2019-03-05 |
| DOI：10.19663/j.issn2095-9869.20190220002 |
| 中文关键词: Loop-mediated isothermal amplification DNA extraction-free Direct gene detection Viable cell Extracellular DNA |
| 英文关键词: Loop-mediated isothermal amplification DNA extraction-free Direct gene detection Viable cell Extracellular DNA |
| 基金项目: |
| 作者 | 单位 | | YANG Qianqian | College of Marine Sciences, Shanghai Ocean University, Shanghai 201306
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071 | | ZHANG Xuzhi | Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071 | | JIANG Xiaoyu | College of Marine Sciences, Shanghai Ocean University, Shanghai 201306
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071 | | LI Yang | Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071 | | ZHAO Jun | Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071 | | HAO Zhihui | School of Chemistry and Pharmaceutical Sciences, Qingdao Agriculture University, Qingdao 266109 | | WANG Pingping | School of Chemistry and Pharmaceutical Sciences, Qingdao Agriculture University, Qingdao 266109 | | QU Keming | Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071 |
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| 中文摘要: |
| When the loop-mediated isothermal amplification (LAMP) assay is used for detecting target genes, DNA extraction is unnecessary in many cases. Simple pretreatment (e.g. heating) is enough to obtain rather sensitive responses. Even test samples without any pretreatment can be used as template. This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies. In this study, using Stx1 gene from E. coli as model, we verified that viable cells, dead cells and extracellular DNA could function as template in the LAMP assay. In the incubation at 63℃, viable bacteria in the LAMP reaction mixture lysed completely within 2 min, providing DNA template for nucleic acid amplification. The Stx1 gene in diluted culture medium, spiked tap water, spiked seawater and real seawater all could be detected, with or without the step of DNA extraction. We found that the complex substances in real sample (e.g. natural seawater) exhibited considerable inhibitory effect on the sensitivity of the LAMP assay. These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring, bio-resource surveys, food safety, etc. in particular those based on environmental DNA. |
| 英文摘要: |
| When the loop-mediated isothermal amplification (LAMP) assay is used for detecting target genes, DNA extraction is unnecessary in many cases. Simple pretreatment (e.g. heating) is enough to obtain rather sensitive responses. Even test samples without any pretreatment can be used as template. This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies. In this study, using Stx1 gene from E. coli as model, we verified that viable cells, dead cells and extracellular DNA could function as template in the LAMP assay. In the incubation at 63℃, viable bacteria in the LAMP reaction mixture lysed completely within 2 min, providing DNA template for nucleic acid amplification. The Stx1 gene in diluted culture medium, spiked tap water, spiked seawater and real seawater all could be detected, with or without the step of DNA extraction. We found that the complex substances in real sample (e.g. natural seawater) exhibited considerable inhibitory effect on the sensitivity of the LAMP assay. These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring, bio-resource surveys, food safety, etc. in particular those based on environmental DNA. |
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