Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid-modified oligonucleotides (LNA) and Mirus Label IT(®) nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5' terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

中文翻译：

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使用锁核酸和微珠阵列技术对有害藻华 (HAB) 进行分子检测。

有害藻华 (HAB) 是沿海水域的严重公共卫生风险。随着 HAB 的强度和频率不断上升，需要新的检测方法来进行可靠的识别。在此，我们开发了一种高通量、多重、珠阵列技术，用于检测甲藻短尾鞭毛虫和 Karenia mikimotoi。该方法将 Luminex 检测系统与两项新技术相结合：锁核酸修饰寡核苷酸 (LNA) 和 Mirus Label IT(®) 核酸技术。为了研究该方法的可行性，我们评估了修饰和未修饰的 LNA 探针的性能，其中扩增子目标用两种不同的策略进行生物素标记：直接化学标记（Mirus Label IT）与酶促末端标记（单一生物素化引物）。结果表明，与未修饰的寡核苷酸 DNA 探针相比，LNA 探针以更好的亲和力与互补单链 DNA 杂交，并显示出更高的荧光强度。当测定在高于 53°C 的温度下进行时，后一种效果更为明显。与酶促 5' 末端标记技术相反，化学标记方法将荧光水平提高了约 83%。使用 LNA 探针和 Mirus Label IT 系统建立的测定检测限范围为 0.05 至 46 个 rRNA 拷贝。这种高通量方法代表了第一个将 Luminex 技术与 LNA 探针和 Mirus Label IT 集成的分子检测策略，