| HOU Jianjun,LAI Hongyan,HUANG Bangqin,CHEN Jixin. 2009. Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry. Acta Oceanologica Sinica, (2):103－114 |
| Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry |
| Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry |
| Received:February 27, 2008 Revised:September 16, 2008 |
| DOI： |
| Key words:oligonucleotide DNA probes Prorocentrum minimum Takayama pulchella fluorescence in situ hybridization flow cytometry |
| 中文关键词: oligonucleotide DNA probes Prorocentrum minimum Takayama pulchella fluorescence in situ hybridization flow cytometry |
| 基金项目:The Fujian Provincial Government of China under contract No. 2005YZ1018; the Xiamen Municipal Government of China under contract No. 3502Z20041059; the China Postdoctoral Science Foundation under contract No. 20060400854; the Open Fund of the State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences under contract No. 2008FB005; the Specialized Research Fund for the Doctoral Program of Higher Education of China under contract 20070504076; the Open Fund of the Key Laboratory of Freshwater Fish Germplasm and Biotechnology of Ministry of Agriculture, Chinese Academy of Fishery Sciences under contract No. LFB20070611; the National Natural Science Foundation of China under contract No. 40576055. |
| Author Name | Affiliation | E-mail | | HOU Jianjun | College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
College of Life Sciences, Hubei Normal University, Huangshi 435002, China
State Key Laboratory of Marine Environmental Science, Environmental Science Research Center, Xiamen University, Xiamen 361005, China
Key Laboratory of Freshwater Fish Germplasm Resources and Biotechnology, Yangtse River Fisheries Research Institute, Jinzhou 434000, China
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China | | | LAI Hongyan | College of Life Sciences, Hubei Normal University, Huangshi 435002, China
Key Laboratory of Freshwater Fish Germplasm Resources and Biotechnology, Yangtse River Fisheries Research Institute, Jinzhou 434000, China | | | HUANG Bangqin | State Key Laboratory of Marine Environmental Science, Environmental Science Research Center, Xiamen University, Xiamen 361005, China | bqhuang@xmu.edu.cn | | CHEN Jixin | State Key Laboratory of Marine Environmental Science, Environmental Science Research Center, Xiamen University, Xiamen 361005, China | |
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| Abstract: |
| Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order:PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples. |
| 中文摘要: |
| Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order:PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples. |
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