Food Quality and Safety Research Group Av. Mestre José Veiga s/n, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330, Braga, Portugal.
Anal Bioanal Chem. 2022 Jul;414(18):5239-5253. doi: 10.1007/s00216-021-03564-5. Epub 2021 Aug 10.
The continuous spread of invasive alien species, as zebra mussel (Dreissena polymorpha), is a major global concern and it is urgent to stop it. Early stages of an invasion are crucial and challenging; however, detection tools based on environmental DNA analysis are promising alternatives. We present an alternative DNA target amplification strategy for signal enhancement followed by dual-mode colorimetric naked eye and optical smartphone analysis for the early detection of zebra mussel environmental DNA. Target amplification was designed based on the nicking endonuclease probe cleavage upon probe and complementary target hybridization. The cleaved/intact probe interacts with DNA-modified nanoparticles for colorimetric detection. We have demonstrated that enzyme amplification strategy enhanced 12-fold the sensitivity by naked eye detection, achieving a detection limit of ~8 nM (4.48×10 copies) in controlled conditions, whereas target in complex environmental samples allowed the detection of 22.5 nM (1.26×10 copies). Competitive assays also showed that the system can discriminate specific zebra mussel DNA sequences from other DNA sequences. Additionally, smartphone analysis for DNA quantification further improved the sensitivity of its detection by 130-fold, more than 2 orders of magnitude, when applied to environmental samples. The limit of detection to 0.17 nM (9.52×10 copies) is based on RGB coordinates, which is especially relevant to monitor early aggregation stages, being more accurate and reducing naked eye detection subjectivity. DNA extracted from zebra mussel meat, zebra mussel contaminated river water, and non-contaminated river water samples were successfully tested. Dual-mode colorimetric detection is useful in field analysis without the need for expensive laboratory equipment.
入侵外来物种（如斑马贻贝（Dreissena polymorpha））的持续传播是一个全球性的主要关注点，急需加以阻止。入侵的早期阶段至关重要且具有挑战性；然而，基于环境 DNA 分析的检测工具是很有前途的替代方法。我们提出了一种替代的 DNA 靶标扩增策略，用于信号增强，随后进行双色比色法和光学智能手机分析，以实现对斑马贻贝环境 DNA 的早期检测。靶标扩增是基于探针和互补靶标杂交时的缺口内切酶探针切割设计的。切割/完整探针与 DNA 修饰的纳米颗粒相互作用以进行比色检测。我们已经证明，酶扩增策略通过肉眼检测将灵敏度提高了 12 倍，在受控条件下实现了约 8 nM（4.48×10 拷贝）的检测限，而在复杂的环境样本中，目标物的检测限为 22.5 nM（1.26×10 拷贝）。竞争性测定还表明，该系统可以从其他 DNA 序列中区分出特定的斑马贻贝 DNA 序列。此外，智能手机分析用于 DNA 定量，当应用于环境样本时，其检测灵敏度进一步提高了 130 倍，超过 2 个数量级。基于 RGB 坐标的检测限为 0.17 nM（9.52×10 拷贝），这对于监测早期聚集阶段特别相关，更加准确且减少了肉眼检测的主观性。从斑马贻贝肉、斑马贻贝污染河水和未污染河水中提取的 DNA 成功进行了测试。无需昂贵的实验室设备，双模式比色检测可用于现场分析。
