No abstract

Background: Toxoplasma gondii is a single-celled parasite commonly found in mammals. Diagnosis of toxoplasmosis largely depends on measurements of the antibody and/or antigen and Toxoplasma-derived DNAs due to the presence of tissue dwelling quiescent cysts and latent infection of the parasite. As a major surface antigen of T.gondii tachyzoites, SAG1 is a key marker for laboratory diagnosis. However, at present, there are no methods available for SAG1 detection using aptamer-based technology.Methods: Recombinant truncated SAG1（r-tSAG1）of Toxoplasma WH3 strain (type Chinese 1) was prokaryotically expressed and subjected to the synthetic oligonucleotide library for selection of nucleic acid aptamer which targets the r-tSAG1, with systematic evolution of ligands by exponential enrichment (SELEX) strategy. The screened specific aptamer-2 was used in direct enzyme-linked aptamer assay (DELAA) to detect native SAG1 obtained from tachyzoite lysates, mouse sera of acute infection, and human sera that had been verified to be positive for ToxoDNAs by PCR amplification. Results: The soluble r-tSAG1 protein was obtained from E.coli lysates by using 0.01M Tris-Cl in PBS, and was purified and identified by immunoblotting, and then labelled with biotin. The screened aptamers were amplified by PCR, followed by DNA sequencing. The results showed that the aptamer-2, with the highest affinity to nSAG1 among the four aptamer candidates, has a higher specificity and sensitivity when used in detection of nSAG1 in the sera of both animals and humans when compared with the commercial Toxoplasma circulating antigen testing kit.Conclusions: A new direct enzyme-linked aptamer assay (DELAA), with aptamer-2 as the recognition probe, was developed for detection of native SAG1 protein secreted by T.gondii. With increased sensitivity and specificity, stability during storage, easy and cheaper production, the aptamer-based technique is considered as a efficient method for the diagnosis of active and reactivated toxoplasmosis.