Safarpour Hossein, Safarnejad Mohammad Reza, Tabatabaie Meisam, Mohsenifar Afshin
Nanosystems Research Team (NRT), Department of Microbial Biotechnology and Biosafety, Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran.
Commun Agric Appl Biol Sci. 2012;77(3):7-13.
The plasmodiophoromycete Polymyxa betae and P. graminis are eukaryotic biotrophic parasites residing in the roots of chenopodiacae and gramineae plants. They are natural transmitting agents of several important plant viruses such as are beet necrotic yellow vein virus (BNYW), beet soil borne mosaic virus (BSBMV), wheat soil-borne mosaic virus (WSBMV). Developing advanced high-throughput diagnostic methods capable of accurate detection of these pathogens could assist with the screening programs and consequently with the development of disease-resistant germplasms. In the present study, a previously developed quantum dots (QDs) FRET-based nano-biosensor was upgraded to a high-throughput version. QDs were functionalized with a specific antibody against the P. betae's specific glutathione-S-transferase (GST) protein. On the other hand, GST was conjugated to Rhodamine dye. Ninety six-well polystyrene plates were used as the detection platform. The mutual affinity of the antigen and the antibody brought the CdTe QDs and rhodamine together close enough to allow the resonance dipole-dipole coupling required for fluorescence resonance energy transfer (FRET) to occur. The immunosensor constructed showed a high sensitivity and specificity of 100%, and was successfully used for high-throughput screening of 96 real samples with consistent results within the course of less than 30 minutes.
根肿菌纲的甜菜多黏菌和禾本科多黏菌是寄生于藜科和禾本科植物根部的真核生物营养型寄生虫。它们是几种重要植物病毒的天然传播媒介，如甜菜坏死黄脉病毒（BNYW）、甜菜土壤传播花叶病毒（BSBMV）、小麦土壤传播花叶病毒（WSBMV）。开发能够准确检测这些病原体的先进高通量诊断方法有助于筛选计划，进而有助于抗病种质的开发。在本研究中，先前开发的基于量子点（QDs）荧光共振能量转移（FRET）的纳米生物传感器被升级为高通量版本。量子点用针对甜菜多黏菌特异性谷胱甘肽-S-转移酶（GST）蛋白的特异性抗体进行功能化。另一方面，GST与罗丹明染料偶联。使用96孔聚苯乙烯板作为检测平台。抗原和抗体的相互亲和力使碲化镉量子点和罗丹明足够靠近，从而使荧光共振能量转移（FRET）所需的共振偶极-偶极耦合得以发生。构建的免疫传感器显示出100%的高灵敏度和特异性，并成功用于96个真实样品的高通量筛选，在不到30分钟的时间内获得了一致的结果。
