Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization

doi:10.1007/s10811-012-9920-3

	Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization; Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization
	Chen, Guo Fu 1,2; Liu, Yang 1; Zhang, Chun Yun 1; Ma, Chao Shuai 1; Zhang, Bao Yu 3; Wang, Guang Ce 3; Xu, Zhong 1; Lu, Dou Ding 4
刊名	JOURNAL OF APPLIED PHYCOLOGY ; JOURNAL OF APPLIED PHYCOLOGY
	2013-08 ; 2013-08
卷号	25 期号:4 页码:1077-1089
关键词	Prorocentrum Prorocentrum LSU rDNA LSU rDNA Probe Probe Fluorescence in situ hybridization Fluorescence in situ hybridization Detection Detection
ISSN号	0921-8971 ; 0921-8971
DOI	10.1007/s10811-012-9920-3 ; 10.1007/s10811-012-9920-3
英文摘要	Prorocentrum is a common dinoflagellate genus along the Chinese seacoast, which frequently causes harmful algal blooms. Efforts to understand and prevent blooms caused by these harmful species require the development of methods for rapid and precise identification and quantification so that an adequate early warning of harmful algal blooms may be given. Here, we report the development and application of rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection of Prorocentrum micans. The hypervariable D1-D2 regions of a large subunit rDNA of a strain isolated from East China Sea identified as P. micans were first sequenced to design species-specific probes. Analysis of sequences identified as P. micans and deposited in GenBank revealed significant base differences among them and phylogenetic analyses revealed multiple clades within the taxon P. micans. Thus, it is likely that more than one taxonomic and genetically distinct entity has been identified as P. micans, if not misidentified. A series of probes were identified to one of these clades and tested for their specificity. Second, whole cell in situ hybridization procedures were established and the optimal probes were screened among the candidate probes. Next, cross-reactivity was performed to test the specificity of the probes and the detection reliability under various culture conditions, including different nutrient levels, temperatures, and light intensities. Finally, an improved protocol for natural samples was applied to the field material. The designed rRNA-targeted probe was specific, showing no cross-reactivity with other microalgae. The optimized detection protocol could be completed within 1.5 h. All target cells were speculated to be identified during all stages of their whole growth cycle under different culture conditions because the difference in fluorescence intensities throughout the experiment was not significant (p > 0.05). The cell densities determined by FISH and light microscopy (LM) were comparable, without any significant difference (p > 0.05) between them. In general, the established FISH probe was promising for specific, rapid, precise detection of a selected set of P. micans in natural samples and served as a good detection model for other Prorocentrum in the future.; Prorocentrum is a common dinoflagellate genus along the Chinese seacoast, which frequently causes harmful algal blooms. Efforts to understand and prevent blooms caused by these harmful species require the development of methods for rapid and precise identification and quantification so that an adequate early warning of harmful algal blooms may be given. Here, we report the development and application of rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection of Prorocentrum micans. The hypervariable D1-D2 regions of a large subunit rDNA of a strain isolated from East China Sea identified as P. micans were first sequenced to design species-specific probes. Analysis of sequences identified as P. micans and deposited in GenBank revealed significant base differences among them and phylogenetic analyses revealed multiple clades within the taxon P. micans. Thus, it is likely that more than one taxonomic and genetically distinct entity has been identified as P. micans, if not misidentified. A series of probes were identified to one of these clades and tested for their specificity. Second, whole cell in situ hybridization procedures were established and the optimal probes were screened among the candidate probes. Next, cross-reactivity was performed to test the specificity of the probes and the detection reliability under various culture conditions, including different nutrient levels, temperatures, and light intensities. Finally, an improved protocol for natural samples was applied to the field material. The designed rRNA-targeted probe was specific, showing no cross-reactivity with other microalgae. The optimized detection protocol could be completed within 1.5 h. All target cells were speculated to be identified during all stages of their whole growth cycle under different culture conditions because the difference in fluorescence intensities throughout the experiment was not significant (p > 0.05). The cell densities determined by FISH and light microscopy (LM) were comparable, without any significant difference (p > 0.05) between them. In general, the established FISH probe was promising for specific, rapid, precise detection of a selected set of P. micans in natural samples and served as a good detection model for other Prorocentrum in the future.
资助项目	Technology and Development Program of Weihai[2010-027] ; Technology and Development Program of Weihai[2010-027]
WOS研究方向	Biotechnology & Applied Microbiology ; Biotechnology & Applied Microbiology ; Marine & Freshwater Biology ; Marine & Freshwater Biology
语种	英语 ; 英语
出版者	SPRINGER ; SPRINGER
WOS记录号	WOS:000321588800018 ; WOS:000321588800018
内容类型	期刊论文
源URL	[http://ir.fio.com.cn/handle/2SI8HI0U/3947]
专题	自然资源部第一海洋研究所
作者单位	1.Harbin Inst Technol Weihai, Coll Oceanol, Weihai 264209, Peoples R China; 2.SOA, Inst Oceanog 1, Qingdao 266061, Peoples R China; 3.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China; 4.SOA, Inst Oceanog 2, Hangzhou 310012, Zhejiang, Peoples R China
推荐引用方式 GB/T 7714	Chen, Guo Fu,Liu, Yang,Zhang, Chun Yun,et al. Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization, Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization[J]. JOURNAL OF APPLIED PHYCOLOGY, JOURNAL OF APPLIED PHYCOLOGY,2013, 2013,25, 25(4):1077-1089, 1077-1089.
APA	Chen, Guo Fu.,Liu, Yang.,Zhang, Chun Yun.,Ma, Chao Shuai.,Zhang, Bao Yu.,...&Lu, Dou Ding.(2013).Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization.JOURNAL OF APPLIED PHYCOLOGY,25(4),1077-1089.
MLA	Chen, Guo Fu,et al."Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization".JOURNAL OF APPLIED PHYCOLOGY 25.4(2013):1077-1089.