In this study, a novel detection method by PCR-based dot chromatography strip (PDCS) is proposed. To investigate the application of PDCS in the detection of harmful microalgae, the internal transcribed spacer sequence of Karlodinium veneficum, one of the most common bloom-forming species, was cloned and sequenced to design and screen specific primers with tag sequences and probes, including gold nanoparticle probe, test probe, and control probe. The PDCS was prepared manually, and PCR amplicons prepared from the genomic DNA of K. veneficum using tagged specific primers were analyzed by PDCS for visual detection of the target species. The resulting test strip showed red spots at the predicted test and control points visible to the naked eyes, showing the successful development of PDCS. This detection technique is independent of expensive experimental equipment (except a DNA thermal cycler for PCR) but requires an aliquot of PCR amplicons mixed with development buffer to apply to the sample pad of PDCS for approximately 10 min to visualize the analytical results. Cross-reactivity test with 21 microalgae, including K. veneficum, showed that the established PDCS technique has excellent specificity. The detection limit of PDCS was 9.13 × 10⁻² ng μL⁻¹ for genomic DNA and 5.3 × 10⁵ cells L ^− 1 for crude DNA extracts of the target alga. In summary, the PDCS with high sensitivity and specificity can be prepared by hand, which is less expensive than traditional strip, thus providing a promising alternative to the detection of K. veneficum in natural samples.

在这项研究中，提出了一种新的基于PCR的点色谱条（PDCS）检测方法。为了研究PDCS在检测有害微藻中的应用，克隆了最常形成水华的物种之一香叶Karlodinium v​​eneficum的内部转录间隔子序列，并进行了测序，以设计和筛选带有标签序列和探针的特异性引物，包括金纳米探针，测试探针和对照探针。手动制备PDCS，并从文氏假单胞菌的基因组DNA制备PCR扩增子使用标记的特异性引物，通过PDCS分析以目测目标物种。所得的测试条在预测的测试点和肉眼可见的控制点上显示红色斑点，表明PDCS成功开发。该检测技术独立于昂贵的实验设备（用于PCR的DNA热循环仪除外），但需要将等分的PCR扩增子与显影缓冲液混合，以将其应用于PDCS的样品垫约10分钟，以使分析结果可视化。与包括K. veneficum在内的21种微藻的交叉反应测试表明，已建立的PDCS技术具有出色的特异性。基因组DNA的PDCS检出限为9.13×10 ^-2  ngμL ^-1，而5.3×10 ⁵L  ^{-  1}细胞，用于目标藻类的粗DNA提取物。总之，可以手工制备具有高灵敏度和特异性的PDCS，它比传统的分离带便宜，因此为检测天然样品中的K. veneficum提供了有希望的替代方法。