Abstract
The dinoflagellate Prorocentrum minimum was successfully detected using loop-mediated isothermal amplification (LAMP) and real-time fluorescence quantitative PCR (RTFQ-PCR). Both specificity and sensitivity testing in the two methods have been validated. In the LAMP assay, the specific ladder-like pattern of bands only appeared in those templates containing P. minimum. The sensitivity of LAMP was tenfold higher than conventional PCR. In RTFQ-PCR assay, only positive amplifications were detected from those samples containing P. minimum. RTFQ-PCR can detect 0.1 cells and 10 pg of DNA within 40 cycles, showing its high sensitivity. Cells could be quantified according to standard curves in agreement with the quantification by standard microscopy counting methods. The LAMP method therefore is appropriate for on-the-spot testing because of its rapidity and simplification, and the RTFQ-PCR is fit for laboratory testing owing to its accurate quantification. The two methods are of significance in forecasting red tides.
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Acknowledgments
This study was supported by the National Basic Research Program of China (973 Program) (Nos. 2010CB428705, 2010CB429005), the Projects of the Science and Technology Commission of Shanghai, China (Nos. 10JC1418600, 062358101), and the Open Project of SOA (State Oceanic Administration of China) Key Laboratory of Integrated Marine Monitoring and Applied Technologies for Harmful Algal Blooms (No. MATHAB20100311), and a special research fund for the national non-profit institutes (East China Sea Fisheries Research Institute) (No. 2007M22). We also thank Kristina Kirshner for English revision of the manuscript.
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Zhang, F., Shi, Y., Jiang, K. et al. Rapid detection and quantification of Prorocentrum minimum by loop-mediated isothermal amplification and real-time fluorescence quantitative PCR. J Appl Phycol 26, 1379–1388 (2014). https://doi.org/10.1007/s10811-013-0163-8
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DOI: https://doi.org/10.1007/s10811-013-0163-8