Centre for Environment Fisheries and Aquaculture Science, Weymouth DT48UB, UK.
Toxins (Basel). 2023 Jun 1;15(6):372. doi: 10.3390/toxins15060372.
The armoured dinoflagellate can be found throughout many of the world's temperate and tropical marine environments. The genus has been studied extensively since approximately half of its members produce a family of potent neurotoxins, collectively called saxitoxin. These compounds represent a significant threat to animal and environmental health. Moreover, the consumption of bivalve molluscs contaminated with saxitoxin poses a threat to human health. The identification of cells collected from sea water samples using light microscopy can provide early warnings of a toxic event, giving harvesters and competent authorities time to implement measures that safeguard consumers. However, this method cannot reliably resolve to a species level and, therefore, is unable to differentiate between toxic and non-toxic variants. The assay outlined in this study uses a quick recombinase polymerase amplification and nanopore sequencing method to first target and amplify a 500 bp fragment of the ribosomal RNA large subunit and then sequence the amplicon so that individual species from the genus can be resolved. The analytical sensitivity and specificity of the assay was assessed using seawater samples spiked with different species. When using a 0.22 µm membrane to capture and resuspend cells, the assay was consistently able to identify a single cell of in 50 mL of seawater. Phylogenetic analysis showed the assay could identify the , , , , , and species from environmental samples, with just the alignment of the reads being sufficient to provide accurate, real-time species identification. By using sequencing data to qualify when the toxic species was present, it was possible to improve the correlation between cell counts and shellfish toxicity from r = 0.386 to r = 0.769 ( ≤ 0.05). Furthermore, a McNemar's paired test performed on qualitative data highlighted no statistical differences between samples confirmed positive or negative for toxic species of by both phylogenetic analysis and real time alignment with the presence or absence of toxins in shellfish. The assay was designed to be deployed in the field for the purposes of in situ testing, which required the development of custom tools and state-of-the-art automation. The assay is rapid and resilient to matrix inhibition, making it suitable as a potential alternative detection method or a complementary one, especially when applying regulatory controls.
装甲甲藻可在世界上许多温带和热带海洋环境中找到。自大约一半的成员产生一组强效神经毒素以来，该属已被广泛研究，统称为石房蛤毒素。这些化合物对动物和环境健康构成重大威胁。此外，食用受石房蛤毒素污染的双壳贝类会对人类健康构成威胁。使用光学显微镜从海水样本中鉴定细胞可以提供有毒事件的早期预警，从而为收获者和主管当局提供时间来实施保护消费者的措施。然而，这种方法不能可靠地解析到种水平，因此无法区分有毒和无毒变体。本研究中概述的测定使用快速重组酶聚合酶扩增和纳米孔测序方法，首先靶向并扩增核糖体 RNA 大亚基的 500 bp 片段，然后对扩增子进行测序，以便可以解析属中的各个物种。使用不同物种的海水样本评估了测定的分析灵敏度和特异性。当使用 0.22 µm 膜捕获和重悬细胞时，该测定能够始终如一的识别 50 mL 海水中的单个 细胞。系统发育分析表明，该测定能够从环境样本中识别 、 、 、 、 和 物种，仅通过读取的比对就足以提供准确的实时物种鉴定。通过使用测序数据来确定有毒 物种的存在时间，可以提高细胞计数与贝类毒性之间的相关性，从 r = 0.386 提高到 r = 0.769（ ≤ 0.05）。此外，对定性数据进行 McNemar 的配对检验表明，通过系统发育分析和实时对齐贝类中是否存在毒素来确认有毒 物种阳性或阴性的样本之间没有统计学差异。该测定是为现场原位测试而设计的，这需要开发定制工具和最先进的自动化技术。该测定快速且对基质抑制具有弹性，非常适合作为潜在的替代检测方法或互补方法，特别是在应用法规控制时。
