HOU Jianjun, LAI Hongyan, HUANG Bangqin, CHEN Jixin. Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry[J]. Acta Oceanologica Sinica, 2009, (2): 103-114.
Citation:
HOU Jianjun, LAI Hongyan, HUANG Bangqin, CHEN Jixin. Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry[J]. Acta Oceanologica Sinica, 2009, (2): 103-114.
HOU Jianjun, LAI Hongyan, HUANG Bangqin, CHEN Jixin. Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry[J]. Acta Oceanologica Sinica, 2009, (2): 103-114.
Citation:
HOU Jianjun, LAI Hongyan, HUANG Bangqin, CHEN Jixin. Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry[J]. Acta Oceanologica Sinica, 2009, (2): 103-114.
Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry
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
2.
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
3.
State Key Laboratory of Marine Environmental Science, Environmental Science Research Center, Xiamen University, Xiamen 361005, China
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.
DownLoad: