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| Direct Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay |
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YANG Qianqian1,2, ZHANG Xuzhi2, JIANG Xiaoyu1,2, LI Yang2, ZHAO Jun2, HAO Zhihui3, WANG Pingping3, QU Keming2
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1.College of Marine Sciences, Shanghai Ocean University, Shanghai 201306;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;3.School of Chemistry and Pharmaceutical Sciences, Qingdao Agriculture University, Qingdao 266109
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| 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. |
| 关键词: Loop-mediated isothermal amplification DNA extraction-free Direct gene detection Viable cell Extracellular DNA |
| DOI： |
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| Direct Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay |
|
YANG Qianqian1,2, ZHANG Xuzhi2, JIANG Xiaoyu1,2, LI Yang2, ZHAO Jun2, HAO Zhihui3, WANG Pingping3, QU Keming2
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|
1.College of Marine Sciences, Shanghai Ocean University, Shanghai 201306;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;3.School of Chemistry and Pharmaceutical Sciences, Qingdao Agriculture University, Qingdao 266109
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| Abstract: |
| 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. |
| Key words: Loop-mediated isothermal amplification DNA extraction-free Direct gene detection Viable cell Extracellular DNA |