Development and Sensitivity Determination of the Toxoplasma gondii Rapid LAMP Assay in Cat Samples
Project no. 22182 · Status: Kapanmış · 2020
Summary
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis in animals and humans. Frequency of infected cats is very important in the transmission of toxoplasmosis to the population. Diagnosis of acute toxoplasmosis in cats is based on microscopic examination of feces or searching for T. gondii DNA by PCR. The frequency of acute toxoplasmosis in cats as definitive hosts in our country was determined in a single study by our study group. After the study in 471 cats, T. gondii-like oocysts were detected in 2 cats by microscopic examination, while the detection of T. gondii DNA by PCR in 66 cats shows that molecular methods are quite sensitive compared to microscopy. However, very expensive equipment and experienced laboratory employees are needed for PCR testing. Therefore, it is predicted that the detection of T. gondii in cats with a sensitive diagnostic test that can be applied in basic laboratory conditions, suitable for field conditions, can reduce the transmission and economic effects of the disease. In this project, it is aimed to develop the Rapid Toxo-LAMP (Loop mediated isothermal amplification) assay, which is a low-cost new molecular assay, which has high specificity and sensitivity for the diagnosis of toxoplasmosis, is suitable for field conditions, has easy and prompt results. For this aim, a specific LAMP assay for Toxoplasma gondii B1 and RE genes has been developed. The topoisomerase (TOPO) vector encoding B1 and RE genes as positive control were used to determine the analytical sensitivity and specificity of the tests. 71 cat samples were confirmed by Real-Time PCR targeting the T. gondii RE gene. Using real-time PCR as a reference method, sensitivities of colorimetric LAMP assays targeting RE and B1 genes were 100% and 97.56% whereas specificities of both assays were 100%. Overall, the colorimetric LAMP RE assay developed in this study brings an advantage in the diagnosis of T. gondii in cat fecal samples since it has higher sensitivity, does not need experienced personnel, and can be applied in basic laboratories or in the field.