Development of a Method for Detecting <i>Alexandrium pacificum</i> Based on the Quantification of <i>sxtA4</i> by Chip-Based Digital PCR
<i>Alexandrium pacificum,</i> 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 <i>A. pacif...
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| Format: | Article |
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MDPI AG
2022-02-01
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| Series: | Toxins |
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| Online Access: | https://www.mdpi.com/2072-6651/14/2/111 |
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| author | Jun-Ho Hyung Jinik Hwang Seung-Joo Moon Eun-Joo Kim Dong-Wook Kim Jaeyeon Park |
| author_facet | Jun-Ho Hyung Jinik Hwang Seung-Joo Moon Eun-Joo Kim Dong-Wook Kim Jaeyeon Park |
| author_sort | Jun-Ho Hyung |
| collection | DOAJ |
| description | <i>Alexandrium pacificum,</i> 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 <i>A. pacificum</i> detection and tested it for monitoring in Jinhae-Masan Bay. Using the sequence of an <i>A. pacificum</i> strain isolated in 2017, species-specific primers targeting <i>sxtA4</i> (a STX biosynthesis-related gene) were designed and used in a dPCR, detecting 2.0 ± 0.24 gene copies per cell of <i>A. pacificum</i>. 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<sup>−1</sup>, 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 <i>sxtA4</i>-based qPCRs. Absolute quantification by chip-based dPCRs targeting <i>sxtA4</i> in <i>A</i>. <i>pacificum</i> exhibits potential as a complementary approach to mouse bioassay PST monitoring for the prevention of toxic blooms. |
| first_indexed | 2024-03-09T20:55:26Z |
| format | Article |
| id | doaj.art-61f6086da786469b80d0bf031f02dc9e |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-03-09T20:55:26Z |
| publishDate | 2022-02-01 |
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| record_format | Article |
| series | Toxins |
| spelling | doaj.art-61f6086da786469b80d0bf031f02dc9e2023-11-23T22:21:33ZengMDPI AGToxins2072-66512022-02-0114211110.3390/toxins14020111Development of a Method for Detecting <i>Alexandrium pacificum</i> Based on the Quantification of <i>sxtA4</i> by Chip-Based Digital PCRJun-Ho Hyung0Jinik Hwang1Seung-Joo Moon2Eun-Joo Kim3Dong-Wook Kim4Jaeyeon Park5Environment and Resource Convergence Center, Advanced Institute of Convergence Technology, Suwon 16229, KoreaWest Sea Fisheries Research Institute, National Institute of Fisheries Science, Incheon 22383, KoreaEnvironment and Resource Convergence Center, Advanced Institute of Convergence Technology, Suwon 16229, KoreaEnvironment and Resource Convergence Center, Advanced Institute of Convergence Technology, Suwon 16229, KoreaFood Safety and Processing Research Division, National Institute of Fisheries Science, Busan 46083, KoreaEnvironment and Resource Convergence Center, Advanced Institute of Convergence Technology, Suwon 16229, Korea<i>Alexandrium pacificum,</i> 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 <i>A. pacificum</i> detection and tested it for monitoring in Jinhae-Masan Bay. Using the sequence of an <i>A. pacificum</i> strain isolated in 2017, species-specific primers targeting <i>sxtA4</i> (a STX biosynthesis-related gene) were designed and used in a dPCR, detecting 2.0 ± 0.24 gene copies per cell of <i>A. pacificum</i>. 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<sup>−1</sup>, 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 <i>sxtA4</i>-based qPCRs. Absolute quantification by chip-based dPCRs targeting <i>sxtA4</i> in <i>A</i>. <i>pacificum</i> exhibits potential as a complementary approach to mouse bioassay PST monitoring for the prevention of toxic blooms.https://www.mdpi.com/2072-6651/14/2/111chip-based digital PCR<i>Alexandrium pacificum</i>paralytic shellfish toxinsaxitoxin biosynthesis geneparalytic shellfish poisoning |
| spellingShingle | Jun-Ho Hyung Jinik Hwang Seung-Joo Moon Eun-Joo Kim Dong-Wook Kim Jaeyeon Park Development of a Method for Detecting <i>Alexandrium pacificum</i> Based on the Quantification of <i>sxtA4</i> by Chip-Based Digital PCR Toxins chip-based digital PCR <i>Alexandrium pacificum</i> paralytic shellfish toxin saxitoxin biosynthesis gene paralytic shellfish poisoning |
| title | Development of a Method for Detecting <i>Alexandrium pacificum</i> Based on the Quantification of <i>sxtA4</i> by Chip-Based Digital PCR |
| title_full | Development of a Method for Detecting <i>Alexandrium pacificum</i> Based on the Quantification of <i>sxtA4</i> by Chip-Based Digital PCR |
| title_fullStr | Development of a Method for Detecting <i>Alexandrium pacificum</i> Based on the Quantification of <i>sxtA4</i> by Chip-Based Digital PCR |
| title_full_unstemmed | Development of a Method for Detecting <i>Alexandrium pacificum</i> Based on the Quantification of <i>sxtA4</i> by Chip-Based Digital PCR |
| title_short | Development of a Method for Detecting <i>Alexandrium pacificum</i> Based on the Quantification of <i>sxtA4</i> by Chip-Based Digital PCR |
| title_sort | development of a method for detecting i alexandrium pacificum i based on the quantification of i sxta4 i by chip based digital pcr |
| topic | chip-based digital PCR <i>Alexandrium pacificum</i> paralytic shellfish toxin saxitoxin biosynthesis gene paralytic shellfish poisoning |
| url | https://www.mdpi.com/2072-6651/14/2/111 |
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