Diaz Mara R, Jacobson James W, Goodwin Kelly D, Dunbar Sherry A, Fell Jack W
Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149.
Limnol Oceanogr Methods. 2010 Jun 1;8:269-284. doi: 10.4319/lom.2010.8.269.
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.
有害藻华（HABs）是沿海水域严重的公共卫生风险。随着有害藻华的强度和频率持续上升，需要新的检测方法来进行可靠识别。在此，我们开发了一种用于检测短裸甲藻和米氏凯伦藻的高通量、多重、微珠阵列技术。该方法将Luminex检测系统与两项新技术相结合：锁核酸修饰的寡核苷酸（LNA）和Mirus Label IT®核酸技术。为研究该方法的可行性，我们用两种不同策略对生物素标记的扩增子靶标评估了修饰和未修饰的LNA探针的性能：直接化学标记（Mirus Label IT）与酶促末端标记（单生物素化引物）。结果表明，LNA探针与互补单链DNA杂交时具有更好的亲和力，并且比未修饰的寡核苷酸DNA探针显示出更高的荧光强度。当检测在53℃以上的温度下进行时，后一种效果更为明显。与酶促5'末端标记技术相反，化学标记方法使荧光水平提高了约83%。用LNA探针和Mirus Label IT系统确定该检测方法的检测限为0.05至46个rRNA拷贝。这种高通量方法是将Luminex技术与LNA探针和Mirus Label IT整合的首个分子检测策略，可适用于检测其他有害藻华，非常适合在藻华前期和爆发期监测赤潮。
