Department of Biotechnology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran.
PLoS One. 2024 Jul 30;19(7):e0293561. doi: 10.1371/journal.pone.0293561. eCollection 2024.
Bovine viral diarrhea virus (BVDV) is the cause of bovine viral diarrhea disease, one of the most economically important livestock diseases worldwide. The majority of BVD disease control programs rely on the detection and then elimination of persistent infection (PI) cattle, as the continuing source of disease. The main purpose of this study was to design and develop an accurate G-quadruplex-based aptasensor for rapid and simple detection of BVDV-1. In this work, we utilized in silico techniques to design a G-quadruplex aptamer specific for the detection of BVDV-1. Also, the rationally designed aptamer was validated experimentally and was used for developing a colorimetric biosensor based on an aptamer-gold nanoparticle system. Firstly, a pool of G-quadruplex forming ssDNA sequences was constructed. Then, based on the stability score in secondary and tertiary structures and molecular docking score, an aptamer (Apt31) was selected. In the experimental part, gold nanoparticles (AuNPs) with an average particle size of 31.7 nm were synthesized and electrostatically linked with the Apt31. The colorimetric test showed that salt-induced color change of AuNPs from red to purple-blue occurs only in the presence of BVDV-Apt31 complex, after 20 min. These results approved the specificity of Apt31 for BVDV. Furthermore, our biosensor could detect the virus at as low as 0.27 copies/ml, which is an acceptable value in comparison to the qPCR method. The specificity of the aptasensor was confirmed through cross-reactivity testing, while its selectivity was confirmed through plasma testing. The sample analysis showed 90% precision and 94% accuracy. It was concluded that the biosensor was adequately sensitive and specific for the detection of BVDV in plasma samples and could be used as a simple and rapid method on the farm.
牛病毒性腹泻病毒（BVDV）是牛病毒性腹泻病的病原体，是全球最重要的经济动物疾病之一。大多数 BVD 疾病控制计划依赖于检测和消除持续性感染（PI）牛，因为它们是疾病的持续来源。本研究的主要目的是设计和开发一种基于 G-四链体的准确适配体传感器，用于快速、简单地检测 BVDV-1。在这项工作中，我们利用计算机技术设计了一种针对 BVDV-1 检测的 G-四链体适配体。此外，还对合理设计的适配体进行了实验验证，并利用适配体-金纳米粒子系统开发了一种比色生物传感器。首先，构建了一组 G-四链体形成的 ssDNA 序列。然后，根据二级和三级结构的稳定性评分和分子对接评分，选择了一个适配体（Apt31）。在实验部分，合成了平均粒径为 31.7nm 的金纳米粒子（AuNPs），并通过静电作用与 Apt31 结合。比色试验表明，只有在存在 BVDV-Apt31 复合物的情况下，盐诱导的 AuNPs 颜色从红色变为紫色-蓝色才会发生，这一过程在 20 分钟后完成。这些结果证明了 Apt31 对 BVDV 的特异性。此外，我们的生物传感器可以检测到低至 0.27 拷贝/ml 的病毒，与 qPCR 方法相比，这是一个可接受的值。通过交叉反应测试验证了适配体传感器的特异性，通过血浆测试验证了其选择性。样品分析显示 90%的精度和 94%的准确率。结论是，该生物传感器对血浆样品中的 BVDV 具有足够的敏感性和特异性，可用于农场的简单快速检测。
