Dataset Information

Development of a Method for Detecting Alexandrium pacificum Based on the Quantification of sxtA4 by Chip-Based Digital PCR.

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.

SUBMITTER: Hyung JH

PROVIDER: S-EPMC8877084 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Development of a Method for Detecting Alexandrium pacificum Based on the Quantification of sxtA4 by Chip-Based Digital PCR.

Hyung Jun-Ho JH Hwang Jinik J Moon Seung-Joo SJ Kim Eun-Joo EJ Kim Dong-Wook DW Park Jaeyeon J

Toxins 20220202 2

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 gen ...[more]

PMID: 35202138

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Project description:Paralytic shellfish poisoning (PSP) is a human foodborne syndrome caused by the consumption of shellfish that accumulate paralytic shellfish toxins (PSTs, saxitoxin group). In PST-producing dinoflagellates such as Alexandrium spp., toxin synthesis is encoded in the nuclear genome via a gene cluster (sxt). Toxin production is supposedly associated with the presence of a 4th domain in the sxtA gene (sxtA4), one of the core genes of the PST gene cluster. It is postulated that gene expression in dinoflagellates is partially constitutive, with both transcriptional and post-transcriptional processes potentially co-occurring. Therefore, gene structure and expression mode are two important features to explore in order to fully understand toxin production processes in dinoflagellates. In this study, we determined the intracellular toxin contents of twenty European Alexandrium minutum and Alexandrium pacificum strains that we compared with their genome size and sxtA4 gene copy numbers. We observed a significant correlation between the sxtA4 gene copy number and toxin content, as well as a moderate positive correlation between the sxtA4 gene copy number and genome size. The 18 toxic strains had several sxtA4 gene copies (9-187), whereas only one copy was found in the two observed non-toxin producing strains. Exploration of allelic frequencies and expression of sxtA4 mRNA in 11 A. minutum strains showed both a differential expression and specific allelic forms in the non-toxic strains compared with the toxic ones. Also, the toxic strains exhibited a polymorphic sxtA4 mRNA sequence between strains and between gene copies within strains. Finally, our study supported the hypothesis of a genetic determinism of toxin synthesis (i.e., the existence of several genetic isoforms of the sxtA4 gene and their copy numbers), and was also consistent with the hypothesis that constitutive gene expression and moderation by transcriptional and post-transcriptional regulation mechanisms are the cause of the observed variability in the production of toxins by A. minutum.

Dataset Information

Development of a Method for Detecting Alexandrium pacificum Based on the Quantification of sxtA4 by Chip-Based Digital PCR.

Publications

Development of a Method for Detecting <i>Alexandrium pacificum</i> Based on the Quantification of <i>sxtA4</i> by Chip-Based Digital PCR.

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