구.농수식품
전남대학교, 해양교란유해생물연구센터
Abstract
Hyun-Gwan Leea,b, Hye Mi Kima,b, Juhee Mina,b, Chungoo Parkb,c, Hae Jin Jeongd, Kitack Leee, Kwang Young Kima,b,*
a Department of Oceanography, College of Natural Sciences, Chonnam National University, Gwangju 61186, Republic of Korea
b Marine Ecosystem Disturbing and Harmful Organisms (MEDHO) Research Center, Gwangju 61186, Republic of Korea
c School of Biological Sciences and Technology, College of Natural Sciences, Chonnam National University, Gwangju 61186, Republic of Korea
d School of Earth and Environmental Sciences, Seoul National University, Seoul 08826, Republic of Korea
e School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang 37673, Republic of Korea
*Corresponding author : Kwang Young Kim
Abstract
A ubiquitous dinoflagellate, Alexandrium, produces paralytic shellfish toxin (PST), and its outbreaks have negative impacts on aquaculture, fisheries, human health, and the marine ecosystem. To minimize such damages, a routine monitoring program of toxic species must be implemented with a suitable analytical technique for their identification and quantification. However, the taxonomic identification and cell quantification of Alexandrium species based on their external morphology under a light microscope, or by using conventional molecular approaches have limited sensitivity and reproducibility. To address these challenges, we have developed an advanced protocol using droplet-digital PCR (ddPCR) for the discrimination and enumeration of three co-occurring Alexandrium species (A. affine, A. catenella, and A. pacificum) in environmental samples. Copies of species-specific internal transcribed spacer (ITS) per cell, which were calculated from environmental samples spiked with various numbers of culture cells, were used to estimate the abundance of species in the field samples. There were no significant differences in ITS copies estimated by the digital PCR assay between environmental samples from different localities, spiked artificially with a consistent number of cells from Alexandrium cultures. This sensitive assay was applied to determine the abundance and vertical distribution of those populations in the southern coastal waters of Korea. In spring, A. catenella was the dominant species, followed by the non-toxic A. affine in summers. A novel digital PCR assay can also be used to monitor other harmful marine protists that require high sample throughput and low detection limit with high accuracy and precision.
Abbreviations
HAB, harmful algal bloom; ITS, internal transcribed spacer; PCR, polymerase chain reaction; ddPCR, droplet-digital PCR; qPCR, quantitative real-time polymerase chain reaction
Keywords
Abundance; Alexandrium spp.; ddPCR; Identification; ITS copy number; Paralytic shellfish toxin (PST); Quantification
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