Development of a lateral flow-based diagnostic kit for the diagnosis of Toxoplasma gondii in cats

Project no. 22468 · Status: Kapanmış · 2021

Summary

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that infects humans and animals and causes toxoplasmosis. It is estimated that 1/3 of the human population in the world is infected with this parasite. Stray cats, which are in frequent contact with humans, have an important role in the transmission of T. gondii to humans and other animals. Infected cats shed infective forms called oocysts, which are infectious to humans and animals, to the environment in large numbers for approximately 20 days. For this reason, it is important for both veterinary and medical to check stray and house cats for T. gondii at certain periods. In this project, it was aimed to develop a new lateral flow-based diagnostic kit for the diagnosis of toxoplasmosis in cats. During the development of this diagnostic kit, the performance of oligonucleotides targeting AF146527.1 gene, and GRA6 and GRA7 peptides selected using immunoinformatic methods as well as the recombinant GRA1 protein were analyzed. During the development of the lateral flow test, cat serum samples known to be positive and negative by ELISA using T. gondii lysate antigen and ELISA using recombinant GRA1 antigen were used. T. gondii DNA positive samples were detected by real time PCR. According to the results obtained, recombinant GRA1 protein performance was found to be better than GRA6 and GRA7 peptides in detecting anti-T. gondii antibodies. When ELISA using lysate antigen or recombinant GRA1 antigen was accepted as the reference test, the sensitivity and specificity of the lateral flow test using recombinant GRA1 antigen were determined as 90% and 100%, respectively. When the positive serum samples were classified according to their optical density (OD) values in recombinant GRA1 ELISA, all of the serum samples which have an OD of >1000 by the ELISA using recombinant GRA1 antigen (n=4) were found positive by lateral flow test. Similarly, all positive sera (n=6) which have an OD of 500<OD<1000 by ELISA using recombinant GRA1 antigen were found positive by lateral flow test. On the other hand, 85% of positive serum samples (n=20) which have OD<500 by ELISA test using recombinant GRA1 antigen could be found positive with the lateral flow test. The performance of the lateral flow test targeting the T. gondii AF146527.1 gene was found to be inadequate. As a result, a lateral flow-based test that can be easily used by veterinarians, gives fast results, and has a sensitivity and specificity of 90% and 100%, respectively, has been developed that can detect anti-T. gondii antibodies in cats. In addition, the sensitivity of the developed lateral flow test reached 100% in serum samples with an OD value of >500 by ELISA.

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