Wang Lijun, Wang Ronghui, Wang Hong, Slavik Michael, Wei Hua, Li Yanbin
College of Food and Biological Engineering, Xihua University, Chengdu 610039, China; Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA.
Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA.
Anal Biochem. 2017 Sep 15;533:34-40. doi: 10.1016/j.ab.2017.06.010. Epub 2017 Jun 20.
Aptamers are single-stranded oligonucleotide ligands that can bind to targets with high affinity and specificity. They have been widely studied in the field of diagnostics as alternatives to antibodies due to their favorable features such as easy labeling, temperature tolerance, lower cost and recognition of a wide variety of targets. In this study, an aptamer-based PCR method coupled with magnetic immunoseparation was developed to detect S. Typhimurium from ground turkey. Firstly, biotinylated polyclonal anti-S. Typhimurium antibody was immobilized on streptavidin-coated magnetic nanobeads to capture S. Typhimurium. Secondly, the aptamers were added and bound to the surface of S. Typhimurium after blocking the magnetic nanobeads with short ssDNA. Finally, the aptamers were released by heating and amplified by PCR. After optimization, this assay was able to detect 10 CFU/mL of S. Typhimurium in pure culture, and 10 CFU/mL of S. Typhimurium in ground turkey. This study demonstrated the feasibility and application of an aptamer-based PCR method coupled with magnetic immunoseparation for sensitive detection of S. Typhimurium in ground turkey.
适体是一种单链寡核苷酸配体，能够以高亲和力和特异性与靶标结合。由于其易于标记、耐温、成本较低以及能够识别多种靶标等有利特性，它们在诊断领域作为抗体的替代品受到了广泛研究。在本研究中，开发了一种基于适体的聚合酶链反应（PCR）方法，并结合磁性免疫分离技术，用于检测火鸡碎肉中的鼠伤寒沙门氏菌。首先，将生物素化的抗鼠伤寒沙门氏菌多克隆抗体固定在链霉亲和素包被的磁性纳米珠上，以捕获鼠伤寒沙门氏菌。其次，在用短链单链DNA封闭磁性纳米珠后，加入适体并使其与鼠伤寒沙门氏菌表面结合。最后，通过加热释放适体，并进行PCR扩增。经过优化，该检测方法能够检测纯培养物中10 CFU/mL的鼠伤寒沙门氏菌，以及火鸡碎肉中10 CFU/mL的鼠伤寒沙门氏菌。本研究证明了基于适体的PCR方法结合磁性免疫分离技术用于灵敏检测火鸡碎肉中鼠伤寒沙门氏菌的可行性和实用性。
