Development of a PNA Probe for Fluorescence In Situ Hybridization Detection of Prorocentrum donghaiense

doi:10.1371/journal.pone.0025527

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	Development of a PNA Probe for Fluorescence In Situ Hybridization Detection of Prorocentrum donghaiense
	Chen, Guofu 1,2,4; Zhang, Chunyu 1; Zhang, Baoyu 2; Wang, Guangce 2,3; Lu, Douding 5; Xu, Zhong 1; Yan, Peishen 1
刊名	PLOS ONE
	2011-10-14
卷号	6 期号:10 页码:e25527
ISSN号	1932-6203
DOI	10.1371/journal.pone.0025527
文献子类	Article
英文摘要	Prorocentrum donghaiense is a common but dominant harmful algal bloom (HAB) species, which is widely distributed along the China Sea coast. Development of methods for rapid and precise identification and quantification is prerequisite for early-stage warning and monitoring of blooms due to P. donghaiense. In this study, sequences representing the partial large subunit rDNA (D1-D2), small subunit rDNA and internal transcribed spacer region (ITS-1, 5.8S rDNA and ITS-2) of P. donghaiense were firstly obtained, and then seven candidate DNA probes were designed for performing fluorescence in situ hybridization (FISH) tests on P. donghaiense. Based on the fluorescent intensity of P. donghaiense cells labeled by the DNA probes, the probe DP0443A displayed the best hybridization performance. Therefore, a PNA probe (PP0443A) analogous to DP0443A was used in the further study. The cells labeled with the PNA probe displayed more intensive green fluorescence than that labeled with its DNA analog. The PNA probe was used to hybridize with thirteen microalgae belonging to five families, i.e., Dinophyceae, Prymnesiophyceae, Raphidophyceae, Chlorophyceae and Bacillariophyceae, and showed no visible cross-reaction. Finally, FISH with the probes PP0443A and DP0443A and light microscopy (LM) analysis aiming at enumerating P. donghaiense cells were performed on the field samples. Statistical comparisons of the cell densities (cells/L) of P. donghaiense in the natural samples determined by FISH and LM were performed using one-way ANOVA and Duncan's multiple comparisons of the means. The P. donghaiense cell densities determined by LM and the PNA probe are remarkably higher than (p < 0.05) that determined by the DNA probe, while no significant difference is observed between LM and the PNA probe. All results suggest that the PNA probe is more sensitive that its DNA analog, and therefore is promising for the monitoring of harmful algal blooms of P. donghaiense in the future.; Prorocentrum donghaiense is a common but dominant harmful algal bloom (HAB) species, which is widely distributed along the China Sea coast. Development of methods for rapid and precise identification and quantification is prerequisite for early-stage warning and monitoring of blooms due to P. donghaiense. In this study, sequences representing the partial large subunit rDNA (D1-D2), small subunit rDNA and internal transcribed spacer region (ITS-1, 5.8S rDNA and ITS-2) of P. donghaiense were firstly obtained, and then seven candidate DNA probes were designed for performing fluorescence in situ hybridization (FISH) tests on P. donghaiense. Based on the fluorescent intensity of P. donghaiense cells labeled by the DNA probes, the probe DP0443A displayed the best hybridization performance. Therefore, a PNA probe (PP0443A) analogous to DP0443A was used in the further study. The cells labeled with the PNA probe displayed more intensive green fluorescence than that labeled with its DNA analog. The PNA probe was used to hybridize with thirteen microalgae belonging to five families, i.e., Dinophyceae, Prymnesiophyceae, Raphidophyceae, Chlorophyceae and Bacillariophyceae, and showed no visible cross-reaction. Finally, FISH with the probes PP0443A and DP0443A and light microscopy (LM) analysis aiming at enumerating P. donghaiense cells were performed on the field samples. Statistical comparisons of the cell densities (cells/L) of P. donghaiense in the natural samples determined by FISH and LM were performed using one-way ANOVA and Duncan's multiple comparisons of the means. The P. donghaiense cell densities determined by LM and the PNA probe are remarkably higher than (p < 0.05) that determined by the DNA probe, while no significant difference is observed between LM and the PNA probe. All results suggest that the PNA probe is more sensitive that its DNA analog, and therefore is promising for the monitoring of harmful algal blooms of P. donghaiense in the future.
学科主题	Life Sciences & Biomedicine - Other Topics
URL标识	查看原文
语种	英语
WOS记录号	WOS:000295981600009
公开日期	2012-07-03
内容类型	期刊论文
源URL	[http://ir.qdio.ac.cn/handle/337002/11836]
专题	海洋研究所_实验海洋生物学重点实验室
作者单位	1.Harbin Inst Technol, State Key Lab Urban Water Resource & Environm, Harbin 150006, Peoples R China 2.Tianjin Univ Sci & Technol, Tianjin Key Lab Marine Resources & Chem, Tianjin, Peoples R China 3.Chinese Acad Sci, Inst Oceanol, Qingdao, Peoples R China 4.SOA, Inst Oceanog 1, Qingdao, Peoples R China 5.SOA, Inst Oceanog 2, Hangzhou, Zhejiang, Peoples R China
推荐引用方式 GB/T 7714	Chen, Guofu,Zhang, Chunyu,Zhang, Baoyu,et al. Development of a PNA Probe for Fluorescence In Situ Hybridization Detection of Prorocentrum donghaiense[J]. PLOS ONE,2011,6(10):e25527.
APA	Chen, Guofu.,Zhang, Chunyu.,Zhang, Baoyu.,Wang, Guangce.,Lu, Douding.,...&Yan, Peishen.(2011).Development of a PNA Probe for Fluorescence In Situ Hybridization Detection of Prorocentrum donghaiense.PLOS ONE,6(10),e25527.
MLA	Chen, Guofu,et al."Development of a PNA Probe for Fluorescence In Situ Hybridization Detection of Prorocentrum donghaiense".PLOS ONE 6.10(2011):e25527.