Rapid detection of Toxoplasma gondii DNA in cat feces using colorimetric loop-mediated isothermal amplification (LAMP) assays targeting RE and B1 genes

Abstract

Toxoplasma gondii is an obligate protozoan parasite that can infect mammals and birds. Cats are the definitive host of T. gondii and have a very important role in transmission of toxoplasmosis due to the shedding of millions of unsporulated oocysts, that become infective in the environment. Since cats play a major key role in the epidemiology of toxoplasmosis, rapid and accurate diagnosis of infected cats has utmost importance. In this study, we developed a novel colorimetric loop mediated isothermal amplification (LAMP) assay detecting T. gondii RE gene and modified a previously developed colorimetric LAMP assay targeting B1 gene to detect T. gondii DNA in cat feces for the first time. The analytical sensitivity of colorimetric LAMP assays was determined using plasmid controls. The clinical sensitivities of both colorimetric LAMPs were determined using cat fecal DNA samples that were confirmed to be positive by two different real-time PCRs in our previous study. According to the results, analytical sensitivities of both assays were 1 copy plasmid/reaction. 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 for experienced personnel, and can be applied in basic laboratories or in the field.