Staggered Target SELEX, a novel approach to isolate non-cross-reactive aptamer for detection of SEA by apta-qPCR

In this study, isolated ssDNA aptamers by ST-SELEX were used for detection of SEA via apta-Real time PCR (apta-qPCR). After in silico analysis of SEA protein with SEE and finding the specific region on the surface of protein, ST-SELEX was carried out in two steps (classical SELEX and Second SELEX). Finally, after isolating high specific aptamers, the apta-qPCR was used for the detection of the SEA. In this technique, poly-clonal antibody against SEA was immobilized on protein G sepharose beads (Ab-PGs). Then, the SEA protein was captured by poly clonal antibody as the target that immobilized on sepharose beads. The isolated aptamers were bound on the surface of SEA protein that captured by Ab-PGs. Finally, the heat-released aptamers were amplified by qPCR.ResultOur investigation showed that the aptamers were generated in vitro by a ten-round selection process based on ST-SELEX procedure with dissociation constant (KD) value 7.44± 0.6 nM and limit of detection (LOD) of 146.67 fM.Discussion and conclusionThe advantage of ST-SELEX compared to other SELEX methods was to select a specific non cross-reactive aptamer against two or more proteins with high sequence homology. These aptamers can be used in sensitive detection methods such as apta-qPCR.
Source: Journal of Biotechnology - Category: Biotechnology Source Type: research