Rapid detection and quantification of Prorocentrum minimum by loop-mediated isothermal amplification and real-time fluorescence quantitative PCR

• DOI: 10.1007/s10811-013-0163-8
• Publication Date: 2013-11-12T07:44:49Z
• Authors: Fengying Zhang; Yanhong Shi; Keji Jiang; Wei Song; Chunyan Ma; Zhaoli Xu; Lingbo Ma
• Keywords: Standard curve; Dinoflagellate; Melting curve analysis

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.