Abstract
Alexandrium pacificum, which produces the paralytic shellfish toxin (PST) saxitoxin (STX), is one of the causative species of paralytic shellfish poisoning outbreaks in coastal areas of Korea. In this study, we developed a chip-based digital PCR (dPCR) method for A. pacificum detection and tested it for monitoring in Jinhae-Masan Bay. Using the sequence of an A. pacificum strain isolated in 2017, species-specific primers targeting sxtA4 (a STX biosynthesis-related gene) were designed and used in a dPCR, detecting 2.0 ± 0.24 gene copies per cell of A. pacificum. Cell abundance in field samples, estimated by a chip-based dPCR, was compared with the PST content, and measured using a mouse bioassay. A comparison with shellfish PST concentrations indicated that cell concentrations above 500 cells L−1, as measured using the dPCR assay, may cause shellfish PST concentrations to exceed the allowed limits for PSTs. Concordance rates between dPCR and PST results were 62.5% overall in 2018–2021, reaching a maximum of 91.7% in 2018–2019. The sensitivity of the dPCR assay was higher than that of microscopy and sxtA4-based qPCRs. Absolute quantification by chip-based dPCRs targeting sxtA4 in A. pacificum exhibits potential as a complementary approach to mouse bioassay PST monitoring for the prevention of toxic blooms.
Highlights
Paralytic shellfish poisoning (PSP) outbreaks derived from harmful algal blooms result in human deaths and economic losses in the fisheries industry worldwide [1,2,3]
Cells isolated from Jinhae-Masan Bay, in the southern coastal waters of Korea, were assigned to the genus Alexandrium based on light microscopy morphological analysis (Figure 1a)
The Masan strain of A. pacificum was taxonomically identified based on a comparative sequence analysis of the large subunit (LSU) and internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) with that of other Alexandrium species deposited in GenBank
Summary
Paralytic shellfish poisoning (PSP) outbreaks derived from harmful algal blooms result in human deaths and economic losses in the fisheries industry worldwide [1,2,3]. A. catenella was thought to be responsible for the algal bloom events in Jinhae-Masan Bay over the past 30 years, Shin et al [12] recently reviewed previous reports and reported that A. pacificum was one of the causative species of these events in the southern coastal waters of Korea, as well as A. catenella, based on resting cysts, the optimal temperature for growth, and phylogenetic analyses. Since this re-evaluation, monitoring of the distribution of A. pacificum in Jinhae-Masan Bay has rarely been reported
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