Toxoplasma gondii is a single-celled parasite commonly found in mammals and birds. Diagnosis of
toxoplasmosis largely depends on measurements of the antibody and/or
antigen and Toxoplasma DNAs due to the presence of tissue dwelling duplicating tachyzoites, or quiescent
cysts in
latent infection of the parasite. As a major
surface antigen of T.gondii tachyzoites, SAG1 is a key molecule for laboratory diagnosis. However, there are no methods available yet for SAG1 detection using aptamer-based technology. Recombinant SAG1 (r-SAG1) of Toxoplasma WH3 strain (type Chinese 1) was expressed in E.coli and subjected to the synthetic
oligonucleotide library for selection of
nucleic acid aptamers which target the r-SAG1
antigen, with systematic evolution of ligands by exponential enrichment (SELEX) strategy. The specific aptamers were screened out and used in direct
enzyme-linked aptamer assay (DELAA) for detection of native SAG1 (n-SAG1) obtained from tachyzoite lysates, mouse sera of acute
infection, and human sera that had been verified for Toxoplasma DNAs by PCR amplification. As results, the soluble r-SAG1
protein was obtained from E.coli lysates by purification and identification with immunoblotting, followed by biotinylation. The selected aptamers were amplified by PCR and
DNA sequencing. The results showed that the aptamer-2, with the highest affinity to n-SAG1 in the sera of animals with minimal difference in the four aptamer candidates, has a high specificity and sensitivity when used in detection of n-SAG1 in the sera of humans when compared with the commercial kit of ELISA for T.gondii circulating
antigen test. We concluded that a new direct
enzyme-linked aptamer assay (DELAA) was developed for the detection of the n-SAG1
protein of T. gondii. With increased sensitivity and specificity, stability, easy and cheap preparation, the aptamer-based technology is considered an efficient method for the diagnosis of active as well as reactivated
toxoplasmosis.