IOCAS-IR  > 海洋生态与环境科学重点实验室
Development of a quantitative PCR for detection and quantification of Prorocentrum donghaiense
Zhang, Chun Yun1,2; Chen, Guo Fu1,2; Zhou, Jin3; Wang, Yuan Yuan1; Lu, Dou Ding4
2016-06-01
发表期刊JOURNAL OF APPLIED PHYCOLOGY
卷号28期号:3页码:1683-1693
文章类型Article
摘要Prorocentrum donghaiense is a dinoflagellate with a high frequency of bloom formation in the East China Sea. These blooms harm coastal ecosystems, marine fisheries, aquatic environments, and public health. Therefore, new and rapid methods that accurately process and specifically detect this alga are crucial to facilitate long-term monitoring or to provide timely warnings of P. donghaiense blooms. We report the development of a quantitative real-time PCR (qPCR) method to identify and detect P. donghaiense. The partial large subunit (LSU) rDNA D1-D2 was cloned and sequenced to design specific amplification primers. The specificity of the primers was tested using regular PCR and fluorescent PCR against a wide range of microalgae widely distributed along the Chinese coast. The qPCR detection protocol was based on two standard curves. Both curves were constructed from standard samples of tenfold serially diluted solutions of the recombinant plasmid containing the LSU D1-D2 fragment and crude DNA extracts with a known number of target cells. A quantitative relationship between the cell numbers and their corresponding plasmid copy numbers was established; this relationship can be used to determine the target cell number of unknown samples in combination with a standard curve that was generated from tenfold-diluted plasmid solutions and the determined C (t) value of target DNA. The effectiveness of the developed protocol was tested with a series of simulated and field samples. The developed qPCR had a detection sensitivity of up to 3.45 cells. The performance of qPCR was not affected by nontarget DNA. The detection test with a series of samples fixed for 40 days showed that qPCR is competent for long-term monitoring programs that require the quantitative analysis of fixative-preserved samples. qPCR can identify the target cells in the field samples within 3-4 h. No significant differences in the quantitative results of the target cells were observed between qPCR and light microscopy. Overall, the established qPCR method is specific, sensitive, rapid, accurate, and promising for the field detection of P. donghaiense in natural samples.
关键词Prorocentrum Donghaiense Lsurdna Quantitative Pcr Detection Dinoflagellate
DOI10.1007/s10811-015-0682-6
收录类别SCI
语种英语
WOS记录号WOS:000375315100024
引用统计
文献类型期刊论文
版本出版稿
条目标识符http://ir.qdio.ac.cn/handle/337002/131057
专题海洋生态与环境科学重点实验室
作者单位1.Harbin Inst Technol Weihai, Sch Marine Sci & Technol, Weihai 264209, Peoples R China
2.Chinese Acad Sci, Inst Oceanol, Key Lab Marine Ecol & Environm Sci, Qingdao 266071, Peoples R China
3.Tsinghua Univ, Div Ocean Sci & Technol, Grad Sch Shenzhen, Shenzhen 518055, Guangdong, Peoples R China
4.SOA, Inst Oceanog 2, Hangzhou 310012, Zhejiang, Peoples R China
推荐引用方式
GB/T 7714
Zhang, Chun Yun,Chen, Guo Fu,Zhou, Jin,et al. Development of a quantitative PCR for detection and quantification of Prorocentrum donghaiense[J]. JOURNAL OF APPLIED PHYCOLOGY,2016,28(3):1683-1693.
APA Zhang, Chun Yun,Chen, Guo Fu,Zhou, Jin,Wang, Yuan Yuan,&Lu, Dou Ding.(2016).Development of a quantitative PCR for detection and quantification of Prorocentrum donghaiense.JOURNAL OF APPLIED PHYCOLOGY,28(3),1683-1693.
MLA Zhang, Chun Yun,et al."Development of a quantitative PCR for detection and quantification of Prorocentrum donghaiense".JOURNAL OF APPLIED PHYCOLOGY 28.3(2016):1683-1693.
条目包含的文件
文件名称/大小 文献类型 版本类型 开放类型 使用许可
Development of a qua(593KB)期刊论文出版稿开放获取CC BY-NC-SA浏览 请求全文
个性服务
推荐该条目
保存到收藏夹
查看访问统计
导出为Endnote文件
谷歌学术
谷歌学术中相似的文章
[Zhang, Chun Yun]的文章
[Chen, Guo Fu]的文章
[Zhou, Jin]的文章
百度学术
百度学术中相似的文章
[Zhang, Chun Yun]的文章
[Chen, Guo Fu]的文章
[Zhou, Jin]的文章
必应学术
必应学术中相似的文章
[Zhang, Chun Yun]的文章
[Chen, Guo Fu]的文章
[Zhou, Jin]的文章
相关权益政策
暂无数据
收藏/分享
文件名: Development of a quantitative PCR for detection and quantification of Prorocentrum donghaiense.pdf
格式: Adobe PDF
所有评论 (0)
暂无评论
 

除非特别说明,本系统中所有内容都受版权保护,并保留所有权利。