背景:由于其广泛应用,原吡虫酮已成为监测和采样中检出率较高的农药之一。因此,因此,有必要建立一种快速、高效的检测原吡喃酮的方法。然而,制约丙咪啶酮快速检测方法发展的一个重要瓶颈是其特异性识别元素的报道很少。在这项工作中,Capture-SELEX和post-SELEX用于适体筛选,和获得的适体用于构建基于适体的侧流测定(LFA)。
结果:首先,通过Capture-SELEX获得了一种针对原吡喃酮的特异性适体Seq15,其解离常数(Kd)为24.22nM。其次,后SELEX用于分析和修改Seq15以提高其性能,截短的序列Seq15-2的Kd为21.28nM。除此之外,通过后SELEX获得广泛特异性适体Seq17-1。Seq17-1可以广泛识别双卡肟杀真菌剂(丙咪啶酮,异丙二酮,氯唑酸盐,敌敌龙和长春唑啉)及其代谢衍生物(3,5-二氯苯胺)。最后,构建了基于特定适体的原吡喃酮LFA,检测限(LOD)为0.79ng/mL。同时,双卡肟杀菌剂及其代谢衍生物的LODs分别为0.62、0.64、0.71、0.69、0.64和0.66ng/mL,分别。上述LFA具有高度特异性和稳定性,并已成功用于蔬菜样品的检测。
结论:在Capture-SELEX和Post-SELEX的组合下,本研究不仅为快速检测丙咪啶酮提供了特异性识别元件,同时也为发现宽特异性适体提供了新的思路。结合广泛特异性初级检测和单特异性定量,已经开发了一种基于适体的复合LFA检测平台,显著提高了检测效率。
BACKGROUND: Due to its wide application, procymidone has become one of the pesticides with high detection rates in supervision and sampling. Therefore, it is necessary to establish a rapid and efficient method for the detection of procymidone. However, an important bottleneck restricting the development of rapid detection methods of procymidone is that its specific recognition elements are rarely reported. In this work, Capture-SELEX and post-SELEX were used in aptamer screening, and the obtained aptamers were used to construct an aptamer-based lateral flow assay (LFA).
RESULTS: Firstly, a specific aptamer Seq15 was obtained for procymidone by Capture-SELEX, and its dissociation constant (Kd) was 24.22 nM. Secondly, post-SELEX was used to analyze and modify Seq15 to improve its performance, and the Kd of the truncated sequence Seq15-2 was 21.28 nM. In addition to this, the broad-specificity aptamer Seq17-1 was obtained via post-SELEX. Seq17-1 could broadly recognize dicarboximide fungicides (procymidone, iprodione, chlozolinate, dimethachlon and vinclozolin) and their metabolic derivative (3,5-dichloroaniline). Finally, the specific aptamer-based LFA of procymidone was constructed, and the limit of detection (LOD) was 0.79 ng/mL. Meanwhile, the LODs of dicarboximide fungicides and their metabolic derivative were 0.62, 0.64, 0.71, 0.69, 0.64 and 0.66 ng/mL, respectively. The above LFAs were highly specific and stable, and had been successfully used for the detection of vegetable samples.
CONCLUSIONS: Under the combination of Capture-SELEX and Post-SELEX, this study not only provides specific recognition elements for rapid detection of procymidone, but also provides new ideas for the discovery of broad-specificity aptamers. Combining broad-specificity primary detection and single-specificity quantification, a composite aptamer-based LFA detection platform has been developed, which significantly improves detection efficiency.