Greenfield Dianne I, Coyne Kathryn J
Advanced Science Research Center at the Graduate Center, City University of New York, New York, NY, USA.
School of Earth and Environmental Sciences, Queens College, City University of New York, New York, NY, USA.
Environ Manage. 2025 Jun;75(6):1589-1601. doi: 10.1007/s00267-025-02156-8. Epub 2025 Apr 8.
Monitoring for harmful algal blooms (HABs) in aquatic environments is commonly aided by light microscopy, though molecular-based approaches can expedite species detection, cell quantification, and therefore early warnings for management responses. Two methods, quantitative polymerase chain reaction (qPCR) and sandwich hybridization assay (SHA), are increasingly used for HAB monitoring, but they differ in terms of protocols, genetic targets, equipment/supplies, and other considerations. This presents a challenge to end-users when selecting tool(s) to integrate within HAB surveillance programs. In response, we conducted a multi-year, side-by-side comparison study between qPCR and SHA relative to microscopy for monitoring the raphidophyte Heterosigma akashiwo, a species responsible for fish kills and impaired water quality worldwide. This paper summarizes key findings from a broad suite of side-by-side, laboratory and field tests of H. akashiwo cell quantification by qPCR and SHA. Assay ranges, detection limits, applicability to preserved samples, and physiological conditions (time of day, growth phase, nutrient levels) of cultured H. akashiwo revealed generally strong qPCR-SHA agreement, though qPCR had a wider dynamic range (without homogenate dilution) while SHA displayed a lower detection limit. Both assays yielded excellent agreement with microscopy during cell growth in the laboratory as well as during bloom development in the field. However, qPCR and SHA cell abundance data were less than microscopy during stationary-decline growth and under low nitrate, indicating reduced cellular nucleic acid during senescence and nutrient stress. Pragmatically, both qPCR and SHA are affordable, but qPCR solutions are typically more available commercially than SHA. Study results will be valuable to managers considering methodological options that suit their HAB monitoring needs.
在水生环境中监测有害藻华（HABs）通常借助光学显微镜，不过基于分子的方法能够加快物种检测、细胞定量，从而实现管理应对的早期预警。定量聚合酶链反应（qPCR）和夹心杂交分析（SHA）这两种方法越来越多地用于有害藻华监测，但它们在实验方案、基因靶点、设备/耗材及其他方面存在差异。这给终端用户在选择纳入有害藻华监测计划的工具时带来了挑战。为此，我们针对监测全球范围内导致鱼类死亡和水质受损的针胞藻赤潮异弯藻，开展了一项qPCR和SHA相对于显微镜的多年并行比较研究。本文总结了通过qPCR和SHA对赤潮异弯藻细胞进行定量的一系列广泛的并行实验室和现场测试的关键发现。对培养的赤潮异弯藻的检测范围、检测限、对保存样本的适用性以及生理条件（一天中的时间、生长阶段、营养水平）的研究表明，qPCR和SHA总体上具有较强的一致性，不过qPCR具有更宽的动态范围（无需匀浆稀释），而SHA的检测限更低。在实验室细胞生长期间以及现场藻华发展期间，两种检测方法与显微镜结果都具有很好的一致性。然而，在稳定下降生长阶段和低硝酸盐条件下，qPCR和SHA的细胞丰度数据低于显微镜检测结果，表明在衰老和营养胁迫期间细胞核酸减少。实际上，qPCR和SHA成本都不高，但qPCR解决方案在商业上通常比SHA更容易获得。研究结果对于考虑适合其有害藻华监测需求的方法选项的管理人员将具有重要价值。
