基于等温扩增反应快速检测水中蓝藻总量的方法研究

Harmful algal blooms, which induced by water eutrophication, have brought substantial risk to human beings and nature in recent years. In order to alleviate these adverse impacts, it is of vital significance to develop a fast and accurate approach for algae monitoring in water. In this project, the cyanobacteria will be took as the model, an isothermal polymerization reaction will be utilized for efficient amplification of the characteristic genes in cyanobacteria, combined with a nanoparticle-based colorimetric quantification technique to establish a visual detection platform for cyanobacteria detection with high sensitivity. The concentration of cyanobacteria can then be deduced after the quantification of the according genes. Comparing to traditional PCR, the isothermal amplification can be performed at a universal temperature, which is much more convenient and simple to operate. Furthermore, the visual detection strategy will be employed to replace conventional quantification methods such as electrophoresis. In this case, the new platform is more suitable for on-field detection. Applying the new technique to the analysis of different water samples, we envision that the critical concentration of cyanobacteria before the outbreak of harmful bloom will be obtained and the according standard will be defined. The completion of this project will contribute to the accurate prediction of water blooms, and more importantly, to the reduction of the undesirable effects caused by algal blooms.

近年来，因水体富营养化引起的的藻类水华给人类和自然界带来了巨大的危害。为了减小水华带来的危害，必须建立一种水体藻类的快速监测预警技术。本项目将以蓝藻为模型，利用核酸等温扩增反应实现蓝藻特征基因的快速高效扩增，同时结合可视化的纳米粒子显色法，构建高灵敏可视化检测平台。通过对蓝藻保守特征基因进行定量，获得其在水中的含量信息。与传统PCR方法相比，等温扩增反应能够在同一温度下进行，操作更为便捷。此外，利用可视化检测代替传统的电泳等定量方法，更利于野外现场检测。利用该检测平台对不同水样进行检测，本项目有望获得水华爆发前水中藻类的临界浓度，并建立相应监测标准。项目的完成将为准确预测水华的爆发并有效降低其危害提供可靠的理论依据。

当前藻类水华在世界上比较普遍存在，蓝藻是引起水华的主要藻类之一。在本研究中，我们通过重组酶聚合酶扩增（RPA）结合侧向层析试纸建立了一种快速的检测蓝藻的方法。在本方法中，蓝藻的DNA通过冻裂法释放。我们通过8种蓝藻种和6种非蓝藻种评估了LF-RPA方法检测蓝藻的有效性和特异性。该方法的检测限为0.01个铜绿微囊藻（FACHB-905）细胞/mL，高于常规的聚合酶链式反应（PCR）的检测限。该方法简单，快速，有效，能实现蓝藻的现场快速检测。