School of Agriculture and Biology, Renji Hospital School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China.
State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Guangzhou 510070, China.
Talanta. 2019 Feb 1;193:199-205. doi: 10.1016/j.talanta.2018.09.084. Epub 2018 Sep 24.
Human noroviruses (HuNoVs) is the primary non-bacterial pathogen causing acute gastroenteritis worldwide. Molecular approaches have been mainly used for detection of HuNoVs. Aptamer-based assay has been also applied for detection of HuNoVs through affinity binding of viral capsid. In a conventional systematic evolution of ligands by exponential enrichment process, the target protein-bound sequences in the library were recovered by complicated process including affinity chromatography, extraction, membrane-filtration or antibody-conjugated magnetic beads. In this study, a novel approach was applied to select aptamers for HuNoVs. The new approach incorporated an in situ capture assay and next generation sequencing (NSG) for selecting the aptamers. P particles of HuNoV (GII.4) were purified and coated on the module to capture sequences that were specifically bound with the protein. The unbound sequences were easily removed by washing. The sequences with high affinity were amplified just in the wells and selected by repeated in situ selection process. From the total of 30,622,226 tested sequences, two aptamers, APTL-1 and APTL-6, were finally selected to incorporate with in situ capture RT-qPCR assay for detection of HuNoVs from clinical samples. The sensitivity of these two aptamers was compared with porcine gastric mucin (PGM) that contains well-known viral receptors, and the reported aptamer APT-M6-2. Both GI and GII HuNoVs could be detected from 5 clinical samples tested. The selected aptamer APTL-1 was comparable to PGM and slightly superior to the reported APTM6-2 aptamer for detection of HuNoVs from clinical samples. The results demonstrated that this in situ target-capture approach for aptamer selection is practicable.
人类诺如病毒（HuNoVs）是全球范围内引起急性胃肠炎的主要非细菌性病原体。分子方法主要用于检测 HuNoVs。基于适配体的检测方法也已应用于通过病毒衣壳的亲和结合来检测 HuNoVs。在常规的指数富集配体系统进化过程中，通过亲和层析、提取、膜过滤或抗体偶联磁珠等复杂过程回收文库中与靶蛋白结合的序列。在这项研究中，应用了一种新的方法来选择针对 HuNoVs 的适配体。该新方法结合了原位捕获测定法和下一代测序（NSG）来选择适配体。HuNoV（GII.4）的 P 粒子被纯化并涂覆在模块上，以捕获与蛋白质特异性结合的序列。通过洗涤可轻松去除未结合的序列。高亲和力的序列仅在孔中扩增，并通过重复原位选择过程进行选择。从总共 30,622,226 个测试序列中，最终选择了两个适配体，APTL-1 和 APTL-6，将其纳入原位捕获 RT-qPCR 测定法中，用于检测来自临床样本的 HuNoVs。这两个适配体的灵敏度与含有已知病毒受体的猪胃粘蛋白（PGM）以及报道的适配体 APT-M6-2 进行了比较。从测试的 5 个临床样本中均可检测到 GI 和 GII HuNoVs。选择的适配体 APTL-1 与 PGM 相当，略优于报道的 APTM6-2 适配体，可用于检测来自临床样本的 HuNoVs。结果表明，这种用于适配体选择的原位靶标捕获方法是可行的。
