Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry
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.
英文摘要:
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.
Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry.pdf（985KB）
1.Xiamen Univ, Environm Sci Res Ctr, State Key Lab Marine Environm Sci, Xiamen 361005, Peoples R China 2.Huazhong Agr Univ, Coll Fisheries, Wuhan 430070, Peoples R China 3.Hubei Normal Univ, Coll Life Sci, Huangshi 435002, Peoples R China 4.Yangtse River Fisheries Res Inst, Key Lab Freshwater Fish Germplasm Resources & Bio, Jinzhou 434000, Peoples R China 5.Chinese Acad Sci, State Key Lab Freshwater Ecol & Biotechnol, Inst Hydrobiol, Wuhan 430072, Peoples R China
Recommended 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,ACTA OCEANOLOGICA SINICA,2009,28(2):103-114
Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry.pdf
