Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
| Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization | |
Chen, Guo Fu1,2; Liu, Yang1 ; Zhang, Chun Yun1; Ma, Chao Shuai1; Zhang, Bao Yu3; Wang, Guang Ce3; Xu, Zhong1; Lu, Dou Ding4; Zhang, CY
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| 2013-08-01 | |
| 发表期刊 | JOURNAL OF APPLIED PHYCOLOGY
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| ISSN | 0921-8971 |
| 卷号 | 25期号:4页码:1077-1089 |
| 文章类型 | Article |
| 摘要 | Prorocentrum is a common dinoflagellate genus along the Chinese seacoast, which frequently causes harmful algal blooms. Efforts to understand and prevent blooms caused by these harmful species require the development of methods for rapid and precise identification and quantification so that an adequate early warning of harmful algal blooms may be given. Here, we report the development and application of rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection of Prorocentrum micans. The hypervariable D1-D2 regions of a large subunit rDNA of a strain isolated from East China Sea identified as P. micans were first sequenced to design species-specific probes. Analysis of sequences identified as P. micans and deposited in GenBank revealed significant base differences among them and phylogenetic analyses revealed multiple clades within the taxon P. micans. Thus, it is likely that more than one taxonomic and genetically distinct entity has been identified as P. micans, if not misidentified. A series of probes were identified to one of these clades and tested for their specificity. Second, whole cell in situ hybridization procedures were established and the optimal probes were screened among the candidate probes. Next, cross-reactivity was performed to test the specificity of the probes and the detection reliability under various culture conditions, including different nutrient levels, temperatures, and light intensities. Finally, an improved protocol for natural samples was applied to the field material. The designed rRNA-targeted probe was specific, showing no cross-reactivity with other microalgae. The optimized detection protocol could be completed within 1.5 h. All target cells were speculated to be identified during all stages of their whole growth cycle under different culture conditions because the difference in fluorescence intensities throughout the experiment was not significant (p > 0.05). The cell densities determined by FISH and light microscopy (LM) were comparable, without any significant difference (p > 0.05) between them. In general, the established FISH probe was promising for specific, rapid, precise detection of a selected set of P. micans in natural samples and served as a good detection model for other Prorocentrum in the future.; Prorocentrum is a common dinoflagellate genus along the Chinese seacoast, which frequently causes harmful algal blooms. Efforts to understand and prevent blooms caused by these harmful species require the development of methods for rapid and precise identification and quantification so that an adequate early warning of harmful algal blooms may be given. Here, we report the development and application of rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection of Prorocentrum micans. The hypervariable D1-D2 regions of a large subunit rDNA of a strain isolated from East China Sea identified as P. micans were first sequenced to design species-specific probes. Analysis of sequences identified as P. micans and deposited in GenBank revealed significant base differences among them and phylogenetic analyses revealed multiple clades within the taxon P. micans. Thus, it is likely that more than one taxonomic and genetically distinct entity has been identified as P. micans, if not misidentified. A series of probes were identified to one of these clades and tested for their specificity. Second, whole cell in situ hybridization procedures were established and the optimal probes were screened among the candidate probes. Next, cross-reactivity was performed to test the specificity of the probes and the detection reliability under various culture conditions, including different nutrient levels, temperatures, and light intensities. Finally, an improved protocol for natural samples was applied to the field material. The designed rRNA-targeted probe was specific, showing no cross-reactivity with other microalgae. The optimized detection protocol could be completed within 1.5 h. All target cells were speculated to be identified during all stages of their whole growth cycle under different culture conditions because the difference in fluorescence intensities throughout the experiment was not significant (p > 0.05). The cell densities determined by FISH and light microscopy (LM) were comparable, without any significant difference (p > 0.05) between them. In general, the established FISH probe was promising for specific, rapid, precise detection of a selected set of P. micans in natural samples and served as a good detection model for other Prorocentrum in the future. |
| 关键词 | Prorocentrum Lsu Rdna Probe Fluorescence In Situ Hybridization Detection |
| 学科领域 | Biotechnology & Applied Microbiology ; Marine & Freshwater Biology |
| DOI | 10.1007/s10811-012-9920-3 |
| URL | 查看原文 |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS研究方向 | Biotechnology & Applied Microbiology ; Marine & Freshwater Biology |
| WOS类目 | Biotechnology & Applied Microbiology ; Marine & Freshwater Biology |
| WOS记录号 | WOS:000321588800018 |
| WOS关键词 | DINOFLAGELLATES ALEXANDRIUM-TAMARENSE ; PSEUDO-NITZSCHIA-AUSTRALIS ; SANDWICH HYBRIDIZATION ; RAPID IDENTIFICATION ; COASTAL WATERS ; BACILLARIOPHYCEAE ; OLIGONUCLEOTIDE ; CATENELLA ; VARIABILITY ; ANTIBODY |
| 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/337002/16560 |
| 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.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China 4.SOA, Inst Oceanog 2, Hangzhou 310012, Zhejiang, Peoples R China |
| 推荐引用方式 GB/T 7714 | Chen, Guo Fu,Liu, Yang,Zhang, Chun Yun,et al. Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization[J]. JOURNAL OF APPLIED PHYCOLOGY,2013,25(4):1077-1089. |
| APA | Chen, Guo Fu.,Liu, Yang.,Zhang, Chun Yun.,Ma, Chao Shuai.,Zhang, Bao Yu.,...&Zhang, CY.(2013).Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization.JOURNAL OF APPLIED PHYCOLOGY,25(4),1077-1089. |
| MLA | Chen, Guo Fu,et al."Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization".JOURNAL OF APPLIED PHYCOLOGY 25.4(2013):1077-1089. |
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| Development of rRNA-（760KB） | 限制开放 | CC BY-NC-SA | 浏览 | |||
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