The Establishment And Application Of Detection Methods Of Three Important Hab Microalgae In The East China Sea With Fluorescent Quantitative PCR

Fluorescent Quantitative Polymerase Chain Reaction (qPCR) is an imperativetechnique in molecular biology. It can detect the amplification process simultaneouslyand calculate the initial quantity of PCR template. Thus, it is widely used in thedetection of mutation and expression of genes, speices identification and quantitativeanalysis. In this study, we applied qPCR technology in the identification andquantification of three important microalgae in the East China Sea-Prorocentrumdonghaiense, Karenia mikimotoi and Skeletonema costatum and the detection methodsof the three microalgae by qPCR were eastablished. Finally, we used these methods inthe identification and quatificaiton of field samples from the East China Sea. Theresults are summarized as below:(1)We designed primers and Taqman probes specific to P. donghaiense and K.mikimotoi respectively on the basis of their ITS (Internal Transcribe Spacer) regionsand prepared recombinant plasmids containing the ITS regions of P. donghaiense, K.mikimotoi and S. costatum, respectively.(2)The detection methods of P. donghaiense and K. mikimotoi by qPCR werefinally established with recombinant plasmids, respectively. The detection method ofSkeletonema costatum by qPCR was optimized and reestablished with its recombinantplasmids. All the methods were determined through verification of laboratory samples.The lower limits of them were4.2×10~2（P. donghaiense）,4.6×10~2(K. mikimotoi)and4.3×10~3(S. costatum) cells respectively, which could meet the needs formonitoring of HABs.(3)We applied the newly eastablished methods in the study of field samples from the East China Sea in April, June and August2011. The results were comparedwith those obtained by light microscopy. It could be concluded that the efficiencies ofthe detection methods by qPCR were silimlar to light microscopy, whichdemonstrated that the qPCR methods were reliable.