Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
| Simultaneous detection of harmful algae by multiple polymerase chain reaction coupled with reverse dot blot hybridization | |
Zhang, Chun Yun1; Chen, Guo Fu1; Liu, Yang1 ; Wang, Yuan Yuan1; Xu, Zhong1; Zhang, Bao Yu2; Wang, Guang Ce2; Chen, GF (reprint author), Harbin Inst Technol Weihai, Sch Marine Sci & Technol, Wenhua West Rd 2, Weihai, Shandong, Peoples R China.
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| 2014-05-01 | |
| 发表期刊 | HARMFUL ALGAE
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| 卷号 | 35页码:9-19 |
| 文章类型 | Article |
| 摘要 | Harmful algal blooms (HABs) caused by microscopic algae present a threat to human health, ecosystem, fishery, tourism, and aquaculture worldwide. HAB warning and monitoring projects require a simple and rapid method for accurate parallel identification of causative algae. This study presents a useful method for simultaneous detection of harmful algae by multiple PCR coupled with reverse dot blot hybridization (MPCRDBH). A variety of probes, including positive, negative, and specific, were first developed by sequencing and consequent sequence analysis of large subunit rDNA D1-D2 from target species and used for specificity test by blot hybridization. The MPCRDBH assay mainly included five steps: (1) microalgal DNA isolation; (2) amplification and labeling of target DNA by multiple PCR; (3) probe tailing and fixation onto positively charged nylon membrane; (4) reverse dot blot hybridization; and (5) hybridization signal recognition by naked eyes. The reverse dot blot hybridization conditions were optimized, and the appropriate parameters were as follows: ultraviolet cross-linking time, 0.5 min; probe density, 2 mu M; Dig-labeled PCR product density, 200 ng; hybridization time and temperature, 2 h and 42 degrees C; and washing time and temperature, 2 x 5 min and 47 degrees C. Sensitivity tests showed that MPCRDBH demonstrated a detection limit of 0.6 cell. MPCRDBH recovered all target species and was not affected by background DNA. MPCRDBH also demonstrated a stable detection performance for fixative (acidic Lugol's solution)-preserved samples over 30 d using simulated field samples. MPCRDBH applicability was assessed and proven effective for parallel detection of target microalgae in the field samples. The developed MPCRDBH exhibited a simple membrane-based DNA array preparation and hybridization signal recognition compared with other current DNA arrays. The assay presented in this study is specific and sensitive for parallel detection of microalgae, with stable performance. Therefore, this assay is promising for field monitoring of natural samples. (C) 2014 Elsevier B.V. All rights reserved. |
| 关键词 | Harmful Algal Blooms Lsu Rdna Multiple Pcr Reverse Dot Blot Hybridization |
| 学科领域 | Marine & Freshwater Biology |
| DOI | 10.1016/j.hal.2014.03.004 |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000336819500002 |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qdio.ac.cn/handle/337002/24166 |
| 专题 | 实验海洋生物学重点实验室 |
| 通讯作者 | Chen, GF (reprint author), Harbin Inst Technol Weihai, Sch Marine Sci & Technol, Wenhua West Rd 2, Weihai, Shandong, Peoples R China. |
| 作者单位 | 1.Harbin Inst Technol Weihai, Coll Oceanol, Weihai 264209, Shandong, Peoples R China 2.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China |
| 推荐引用方式 GB/T 7714 | Zhang, Chun Yun,Chen, Guo Fu,Liu, Yang,et al. Simultaneous detection of harmful algae by multiple polymerase chain reaction coupled with reverse dot blot hybridization[J]. HARMFUL ALGAE,2014,35:9-19. |
| APA | Zhang, Chun Yun.,Chen, Guo Fu.,Liu, Yang.,Wang, Yuan Yuan.,Xu, Zhong.,...&Chen, GF .(2014).Simultaneous detection of harmful algae by multiple polymerase chain reaction coupled with reverse dot blot hybridization.HARMFUL ALGAE,35,9-19. |
| MLA | Zhang, Chun Yun,et al."Simultaneous detection of harmful algae by multiple polymerase chain reaction coupled with reverse dot blot hybridization".HARMFUL ALGAE 35(2014):9-19. |
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