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DOI10.1016/j.hal.2019.01.011
Rapid and sensitive detection method for Karlodinium veneficum by recombinase polymerase amplification coupled with lateral flow dipstick
Fu, Mengqi1; Chen, Guofu1; Zhang, Chunyun1,2; Wang, Yuanyuan1; Sun, Rui1; Zhou, Jin3
发表日期2019
ISSN1568-9883
EISSN1878-1470
卷号84页码:1-9
英文摘要

The dinoflagellate Karlodinium veneficum that is usually present at relatively low cell abundances is a globally-distributed harmful algal bloom-forming species, which negatively affects marine ecosystems, fisheries, and human health. Hence, an efficient detection platform for the rapid and sensitive identification of K. veneficum is highly demanded. In this study, a method referred to as recombinase polymerase amplification coupled with lateral flow dipstick (RPA-LFD) was developed for the rapid detection of K. veneficum. The primers for RPA and the detection probe for LFD were designed to specially target the internal transcribed spacer of K. veneficum by molecular cloning and multiple alignments of the related sequences. The developed RPA can gain an approximately 300 bp specific band from K. veneficum. Successful amplification for RPA could be achieved at a temperature range of 35 degrees C-45 degrees C. RPA for 30 min could produce enough products that could generate clearly visible electrophoresis bands and were adequate for subsequent LFD analysis. The RPA products can be visually detected by the naked eyes through an LFD after an automatic chromatography for 5 min at room temperature. The developed RPA-LFD was exclusively specific for K. veneficum and displayed no cross-reactivity with other algal species that are commonly distributed along the Chinese coast. In addition, the lowest detection limit of RPALFD was 10 ng mu L-1 of genomic DNA and 0.1 cell mL(-1), which was 100-fold sensitive than conventional PCR. In conclusion, the developed RPA-LFD assay in this study can be used as a rapid and sensitive method to monitor K. veneficum in the future.


WOS研究方向Marine & Freshwater Biology
来源期刊HARMFUL ALGAE
文献类型期刊论文
条目标识符http://gcip.llas.ac.cn/handle/2XKMVOVA/89816
作者单位1.Harbin Inst Technol Weihai, Sch Marine Sci & Technol, Wenhua West Rd 2, Weihai 264209, Shandong, Peoples R China;
2.Ningbo Univ, Sch Marine Sci, Ningbo 315211, Zhejiang, Peoples R China;
3.Tsinghua Univ, Grad Sch Shenzhen, Div Ocean Sci & Technol, Shenzhen 518055, Peoples R China
推荐引用方式
GB/T 7714
Fu, Mengqi,Chen, Guofu,Zhang, Chunyun,et al. Rapid and sensitive detection method for Karlodinium veneficum by recombinase polymerase amplification coupled with lateral flow dipstick[J],2019,84:1-9.
APA Fu, Mengqi,Chen, Guofu,Zhang, Chunyun,Wang, Yuanyuan,Sun, Rui,&Zhou, Jin.(2019).Rapid and sensitive detection method for Karlodinium veneficum by recombinase polymerase amplification coupled with lateral flow dipstick.HARMFUL ALGAE,84,1-9.
MLA Fu, Mengqi,et al."Rapid and sensitive detection method for Karlodinium veneficum by recombinase polymerase amplification coupled with lateral flow dipstick".HARMFUL ALGAE 84(2019):1-9.
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