Journal of biotechnology · 2018
Staggered Target SELEX, a novel approach to isolate non-cross-reactive aptamer for detection of SEA by apta-qPCR.
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Sedighian Hamid, Halabian Raheleh, Amani Jafar, Heiat Mohammad, Amin Mohsen, Fooladi Abbas Ali Imani
Аннотация
BACKGROUND AND OBJECTIVE: Aptamers or chemical antibodies are oligonucleotides (DNA or RNA) that are able to bind to various targets with high specificity and affinity such as toxins which are isolated by an in vitro method known as SELEX. To date, there are many SELEX procedures for the isolation of novel aptamers against proteins. However not all modified SELEX are suitable for similar protein based on sequence homology such as staphylococcal enterotoxins. Staphylococcal enterotoxin type A (SEA) is the most prevalent toxin involved in staphylococcal food poisoning (SFP) worldwide. SEA is homologous to Staphylococcal enterotoxin type D (SED) and Staphylococcal enterotoxin type E (SEE) about 50% and 83%, respectively. Here, we have developed Staggered Target SELEX (ST-SELEX) as a novel designed SELEX procedure to acquire specific non-cross-reactive aptamers against SEA as a model protein.
METHODS: 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.
RESULT: Our 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 CONCLUSION: The 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.
METHODS: 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.
RESULT: Our 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 CONCLUSION: The 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.