PDF(Pubmed)
Abstract：
Paralytic shellfish toxins (PSTs) produced by marine dinoflagellates significantly impact shellfish industries worldwide. Early detection on-farm and with minimal training would allow additional time for management decisions to minimize economic losses. Here, we describe and test a standardized workflow based on the detection of sxtA4, an initial gene in the biosynthesis of PSTs. The workflow is simple and inexpensive and does not require a specialized laboratory. It consists of (1) water collection and filtration using a custom gravity sampler, (2) buffer selection for sample preservation and cell lysis for DNA, and (3) an assay based on a region of sxtA, DinoDtec lyophilized quantitative polymerase chain reaction (qPCR) assay. Water samples spiked with Alexandrium catenella showed a cell recovery of >90% when compared to light microscopy counts. The performance of the lysis method (90.3% efficient), Longmire\'s buffer, and the DinoDtec qPCR assay (tested across a range of Alexandrium species (90.7-106.9% efficiency; r2 > 0.99)) was found to be specific, sensitive, and efficient. We tested the application of this workflow weekly from May 2016 to 30th October 2017 to compare the relationship between sxtA4 copies L-1 in seawater and PSTs in mussel tissue (Mytilus galloprovincialis) on-farm and spatially (across multiple sites), effectively demonstrating an ∼2 week early warning of two A. catenella HABs (r = 0.95). Our tool provides an early, accurate, and efficient method for the identification of PST risk in shellfish aquaculture.
摘要：
海洋鞭毛藻产生的麻痹性贝类毒素（PST）极大地影响了全球贝类产业。在农场及早发现并进行最少的培训将为管理决策提供更多时间，以最大程度地减少经济损失。这里,我们描述并测试了基于sxtA4检测的标准化工作流程,sxtA4是PST生物合成中的初始基因.工作流程简单且便宜，不需要专门的实验室。它包括（1）使用自定义重力采样器收集和过滤水，（2）样品保存和细胞裂解DNA的缓冲液选择，和（3）基于sxtA区域的测定，DinoDtec冻干定量聚合酶链反应(qPCR)测定。当与光学显微镜计数相比时,掺有Alexanderiumcatenella的水样显示>90%的细胞回收率。裂解法的性能（90.3%的效率），Longmire的缓冲区,发现DinoDtecqPCR检测（在一系列Alexandrium物种中进行了测试（效率90.7-106.9％；r2>0.99））具有特异性，敏感,高效。我们从2016年5月至2017年10月30日每周测试此工作流程的应用，以比较海水中sxtA4副本L-1与贻贝组织（Mytilusgalloprovincialis）在农场和空间（跨多个地点）中的PST之间的关系。有效地证明了两个A.catenellaHABs的2周预警(r=0.95)。我们的工具提供了一个早期，准确,贝类养殖PST风险识别的有效方法。
