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 desig...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. pulcheUa 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 Mgae in natural samples.展开更多
Toxic cyanobacterial blooms constitute a threat to human safety because Microcystis sp. releases microcystins during growth, and particularly during cell death. Therefore, analysis of toxic and nontoxic Microcystis in...Toxic cyanobacterial blooms constitute a threat to human safety because Microcystis sp. releases microcystins during growth, and particularly during cell death. Therefore, analysis of toxic and nontoxic Microcystis in natural communities is required in order to assess and predict bloom dynamics and toxin production by these organisms. In this study, an analysis combining fluorescence in situ hybridization (FISH) with flow cytometry (FCM) was used to discriminate between toxic and nontoxic Microcystis and also to quantify the percentage of toxic Microcystis present in blooms. The results demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of Microcystis toxin production and for providing an early warning for toxic Microcystis blooms.展开更多
基金The Fujian Provincial Government of China under contract No 2005YZ1018 the Xiamen Municipal Government of China under contract No 3502Z20041059+4 种基金 the China Postdoctoral Science Foundation under contract No 20060400854the 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 LFB20070611the National Natural Science Foundation of China under contract No 40576055
文摘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. pulcheUa 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 Mgae in natural samples.
基金supported by the National Basic Research Program of China (Grant No. 2008CB418000)the National Water Pollution Control and Management Technology Major Project (Grant No. 2009ZX07527- 005)+1 种基金the Natural Science Foundation of China-Yunnan Project (Grant No. U0833604)the Knowledge Innovative Program of the Chinese Academy of Sciences (Grant No. KZCX1-YW-14-1)
文摘Toxic cyanobacterial blooms constitute a threat to human safety because Microcystis sp. releases microcystins during growth, and particularly during cell death. Therefore, analysis of toxic and nontoxic Microcystis in natural communities is required in order to assess and predict bloom dynamics and toxin production by these organisms. In this study, an analysis combining fluorescence in situ hybridization (FISH) with flow cytometry (FCM) was used to discriminate between toxic and nontoxic Microcystis and also to quantify the percentage of toxic Microcystis present in blooms. The results demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of Microcystis toxin production and for providing an early warning for toxic Microcystis blooms.
