Method for quantifying sxtA4 gene from Alexandrium minutum using chip-based digital PCR (dPCR) for estimation toxin level which causing paralytic shellfish poisoning

jin Ik Hwang1, Eun Young Yoon2, Seung Joo Moon3, Jung-rae Rho4 and Jaeyeon Park1, (1)Advanced Institution of Convergence Technology, Suwon, South Korea, (2)Advanced Institutes of Convergence Technology, Environment and Resource Convergence Center, Suwon, South Korea, (3)Kunsan National University, Oceanography, Kunsan, South Korea, (4)Kunsan National University, Kunsan, South Korea
Abstract:
Method for quantifying sxtA4 gene from Alexandrium minutum using chip-based digital PCR (dPCR) for estimation toxin level which causing paralytic shellfish poisoning

Jinik Hwang1, Eun Young Yoon1, Seung Joo Moon2, Jung rae Rho2, Jaeyeon Park1

1Environment and Resource Convergence Center, Advanced Institutes of Convergence Technology, Suwon 16229, Republic of Korea

2Department of Oceanography, Kunsan National University, Jeonbuk 573-701, Republic of Korea

Abstract

Marine dinoflagellates species in genus Alexandrium are known to produce potent neurotoxins called the saxitoxin and this toxin is best-known paralytic shellfish toxin (PST). Ingestion of saxitoxin by humans is usually by consumption of shellfish which contaminated by toxic algae blooms, is responsible for the illness known as paralytic shellfish poisoning (PSP). Early detection by monitoring of the toxin and the STX-producing dinoflagellates can prevent the issues related to harmful algal blooms and human poisoning. However, conventional methods using qPCR for detecting causative species have the limit for precise quantification which affected by PCR inhibitors in seawater samples. Therefore we have developing a new analytical method using chip-based on digital PCR (dPCR), adding aspects of fluorescence-activated sorting for providing absolute quantification of STX-producing dinoflagellates Chip-based digital PCR method were using for quantifing sxtA4, catalytic domains of the sxtA4 gene which is essential for SXT biosynthesis in dinoflagellates. The gene copy number of sxtA4 in Alexandrium minutum cells could be calculated by using the chip-based digital PCR. Estimation of STX at the gene expression levels using dPCR will be useful for predicting and investigating the occurrence of increasing toxicity using seawater samples by detecting the biomass of causative species.

Key word: Quantification Digital PCR, sxtA4 gene, paralytic shellfish toxin (PST), Alexandrium minutum