Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry |
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Authors: | HOU Jianjun LAI Hongyan HUANG Bangqin and CHEN Jixin |
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Institution: | 1
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
2
College of Life Sciences, Hubei Normal University, Huangshi 435002, China
3
State Key Laboratory of Marine Environmental Science, Environmental Science Research Center, Xiamen University, Xiamen 361005, China
4
Key Laboratory of Freshwater Fish Germplasm Resources and Biotechnology, Yangtse River Fisheries Research Institute, Jinzhou 434000, China
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State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China |
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Abstract: | Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were ampli.ed, 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 .uorescence in situ hybridization method using .ow cytometry. Epi.uorescence micrographs showed that these speci.c probes labeled with .uorescein isothiocyanate entered the algal cells and bound to target sequences, and the .uorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were speci.c and e.ective for P. minimum and T. pulchella, without any speci.c binding to other algal species. The hybridization e.ciency of di.erent probes speci.c to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes speci.c to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The di.erent hybridization e.ciency of the DNA probes could also be shown in the .uorescent signals between the labeled and unlabeled cells demonstrated using .ow 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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Keywords: | oligonucleotide DNA probes Prorocentrum minimum Takayama pulchella fluorescence in situ hybridization flow cytometry |
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