Ahn Ji-Young, Lee Kyeong-Ah, Lee Moon-Jong, Sekhon Simranjeet Singh, Rhee Sung-Keun, Cho Sung-Jin, Ko Jung Ho, Lee Lyon, Han Janet, Kim Sang Yong, Min Jiho, Kim Yang-Hoon
School of Biological Sciences, Chungbuk National University 1 Chungdae-Ro, Seowon-Gu, Cheongju 28644, South Korea.
College of Veterinary Medicine, Western University of Health Sciences, Pomona CA 91766, USA.
J Nanosci Nanotechnol. 2018 Mar 1;18(3):1599-1605. doi: 10.1166/jnn.2018.14212.
In this paper, whole-bacteria SELEX (WB-SELEX) strategy was adopted to isolate specific aptamers against Vibrio parahaemolyticus. Round selection for V. parahaemolyticus was conducted 11 rounds, including two negative selection rounds. It was determined through real-time PCR amplification and post-SELEX experiment. The selected aptmers had high binding property and specificity to V. parahaemolyticus. Of 28 aptamers tested, VPCA-apta#1 had the highest binding affinity compared to other aptamer candidates obtained. To detect V. parahaemolyticus, aptamer based SPR biosensor platform was constructed and pathogenic bacteria sensing was conducted in two steps. The first step was to construct 5'-biotinylated VPCA-apta#1 binding probe. The second step was to incubate V. parahaemolyticus and test microbes in functionalized SA sensor chip in parallel. Our platform showed significant activity for detecting and discriminating V. parahaemolyticus from other enteric species such as Escherichia coli, Listeria monocytogenes, Sigella sonnei, and Vibrio fischeri. This is the first report on the use of whole-SELEX to isolate DNA aptamers specific for V. parahaemolyticus. We demonstrated the feasibility of using aptamer platform for the detection of V. parahaemolyticus in various food supplies. It might be used in multiple points of care for diagnosing Vibriosis.
在本文中，采用全细菌SELEX（WB-SELEX）策略来分离针对副溶血性弧菌的特异性适体。对副溶血性弧菌进行了11轮循环筛选，包括两轮阴性筛选。通过实时PCR扩增和SELEX后实验进行测定。所筛选的适体对副溶血性弧菌具有高结合特性和特异性。在测试的28个适体中，与获得的其他适体候选物相比，VPCA-apta#1具有最高的结合亲和力。为了检测副溶血性弧菌，构建了基于适体的SPR生物传感器平台，并分两步进行病原菌传感。第一步是构建5'-生物素化的VPCA-apta#1结合探针。第二步是将副溶血性弧菌和测试微生物并行孵育在功能化的SA传感器芯片中。我们的平台在检测和区分副溶血性弧菌与其他肠道菌种（如大肠杆菌、单核细胞增生李斯特菌、宋内志贺菌和费氏弧菌）方面显示出显著活性。这是关于使用全SELEX分离副溶血性弧菌特异性DNA适体的首次报道。我们证明了使用适体平台检测各种食品供应中副溶血性弧菌的可行性。它可能用于弧菌病诊断的多个护理点。
