Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
| A rapid and sensitive method for field detection of Prorocentrum donghaiense using reverse transcription-coupled loop-mediated isothermal amplification | |
Chen, Guofu1,2; Ma, Chaoshuai1; Zhang, Chunyun1,2; Zhou, Jin3; Wang, Yuanyuan1; Wang, Guangce4; Zhang, Baoyu4; Xu, Zhong1; Lu, Dou Ding5; Zhang, CY
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| 2013-10-01 | |
| 发表期刊 | HARMFUL ALGAE
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| ISSN | 1568-9883 |
| 卷号 | 29页码:31-39 |
| 文章类型 | Article |
| 摘要 | Prorocentrum donghaiense is the most common bloom-forming species in the East China Sea, causing serious damage to regional marine ecosystems, marine fisheries, public health, and aquatic environment. To minimize fisheries losses caused by this harmful alga, a simple and accurate detection method need to be developed to provide adequate early warning for P. donghaiense blooms. In this study, we report the development and application of reverse transcription-coupled loop-mediated isothermal amplification (RT-LAMP) in the field detection of P. donghaiense. The partial large subunit rDNA (LSU D1-D2), small subunit rDNA, and internal transcribed spacers of P. donghaiense were first sequenced to design species-specific amplification primers. Primer set screen showed that the LSU D1-D2 was the best target for LAMP in terms of sensitivity and stability. The amplification conditions including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time, and temperature, were optimized. The specificity of RT-LAMP for P. donghaiense was also confirmed through tests using a few common harmful algae. RT-LAMP targeting RNA exhibited a detection limit of 0.6 cells, which is more sensitive than LAMP and PCR targeting DNA and reverse transcription-polymerase chain reaction (RT-PCR) targeting RNA. Finally, an improved protocol for natural samples was applied to the field material. The optimized detection protocol could be completed within 1 h. In addition, positive RT-LAMP results could be confirmed through the production of white magnesium pyrophosphate precipitate or through mixing the fluorescent dye GeneFinder (TM) with the amplification products. In summary, the established RT-LAMP is specific, sensitive, and rapid method that is promising for the field detection of P. donghaiense. (c) 2013 Elsevier B.V. All rights reserved.; Prorocentrum donghaiense is the most common bloom-forming species in the East China Sea, causing serious damage to regional marine ecosystems, marine fisheries, public health, and aquatic environment. To minimize fisheries losses caused by this harmful alga, a simple and accurate detection method need to be developed to provide adequate early warning for P. donghaiense blooms. In this study, we report the development and application of reverse transcription-coupled loop-mediated isothermal amplification (RT-LAMP) in the field detection of P. donghaiense. The partial large subunit rDNA (LSU D1-D2), small subunit rDNA, and internal transcribed spacers of P. donghaiense were first sequenced to design species-specific amplification primers. Primer set screen showed that the LSU D1-D2 was the best target for LAMP in terms of sensitivity and stability. The amplification conditions including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time, and temperature, were optimized. The specificity of RT-LAMP for P. donghaiense was also confirmed through tests using a few common harmful algae. RT-LAMP targeting RNA exhibited a detection limit of 0.6 cells, which is more sensitive than LAMP and PCR targeting DNA and reverse transcription-polymerase chain reaction (RT-PCR) targeting RNA. Finally, an improved protocol for natural samples was applied to the field material. The optimized detection protocol could be completed within 1 h. In addition, positive RT-LAMP results could be confirmed through the production of white magnesium pyrophosphate precipitate or through mixing the fluorescent dye GeneFinder (TM) with the amplification products. In summary, the established RT-LAMP is specific, sensitive, and rapid method that is promising for the field detection of P. donghaiense. (c) 2013 Elsevier B.V. All rights reserved. |
| 关键词 | Lsu Rdna Prorocentrum Donghaiense Reverse Transcription-coupled Loop-mediated Isothermal Amplification |
| 学科领域 | Marine & Freshwater Biology |
| DOI | 10.1016/j.hal.2013.08.001 |
| URL | 查看原文 |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS研究方向 | Marine & Freshwater Biology |
| WOS类目 | Marine & Freshwater Biology |
| WOS记录号 | WOS:000325741800005 |
| WOS关键词 | EAST CHINA SEA ; SANDWICH HYBRIDIZATION FORMATS ; NUCLEASE PROTECTION ASSAY ; RNA-TARGETED PROBES ; CHAIN-REACTION ; WHOLE-CELL ; DNA ; DINOPHYCEAE ; GENE ; BACILLARIOPHYCEAE |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qdio.ac.cn/handle/337002/16518 |
| 专题 | 实验海洋生物学重点实验室 |
| 通讯作者 | Zhang, CY |
| 作者单位 | 1.Harbin Inst Technol Weihai, Coll Oceanol, Weihai 264209, Peoples R China 2.SOA, Inst Oceanog 1, Qingdao 266061, Peoples R China 3.Tsinghua Univ, Grad Sch Shenzhen, Div Ocean Sci & Technol, Shenzhen 518055, Peoples R China 4.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China 5.SOA, Inst Oceanog 2, Hangzhou 310012, Zhejiang, Peoples R China |
| 推荐引用方式 GB/T 7714 | Chen, Guofu,Ma, Chaoshuai,Zhang, Chunyun,et al. A rapid and sensitive method for field detection of Prorocentrum donghaiense using reverse transcription-coupled loop-mediated isothermal amplification[J]. HARMFUL ALGAE,2013,29:31-39. |
| APA | Chen, Guofu.,Ma, Chaoshuai.,Zhang, Chunyun.,Zhou, Jin.,Wang, Yuanyuan.,...&Zhang, CY.(2013).A rapid and sensitive method for field detection of Prorocentrum donghaiense using reverse transcription-coupled loop-mediated isothermal amplification.HARMFUL ALGAE,29,31-39. |
| MLA | Chen, Guofu,et al."A rapid and sensitive method for field detection of Prorocentrum donghaiense using reverse transcription-coupled loop-mediated isothermal amplification".HARMFUL ALGAE 29(2013):31-39. |
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