Method Verification of Inhouse Real-time Polymerase Chain Reaction for Detection of Toxoplasma gondii

Toxoplasmosis is one of the most common parasitic infection in humans. Serological and molecular methods are used for diagnosis. Molecular methods are becoming increasingly preferred, since they lead to shortening of diagnostic time. In our study, it was aimed to determine Toxoplasma gondii by a cost-effective, quantitative, fast and reliable method without using a commercial kit, and apply method verification. T.gondii strain which was continued by mouse inoculation in our laboratory was used for method verification study. For this purpose DNA extraction was performed using a commercial kit. The limit of detection and, high and low positivity rates were determined by serial dilutions of DNA sample. Accuracy and certainty studies were performed using with TG-F, TG-R primers and TaqMan TG probe for method verification of the test. In the study with serial dilutions of DNA sample, detection limit was determined as 10(-3) dilutions (0.028 copies/reaction). Furthermore 10(-1) dilution (2.8 copies/reaction) was considered as high positive, 10(-2) dilution (0.28 copies/reaction) was considered as low positive and method verification studies were performed. The accuracy of test was determined as 0.62 for high positive samples and 0.14 for low positive samples. CV value of intra-assay certainty was 0.62 for high positive samples and 0.14 for low positive samples, whereas, CV value of inter-assay certainty was calculated as 1.03 for high positive samples and 2.34 for low positive samples. Correlation coefficient was determined as 0.99. The coefficient of variation of inhouse realtime PCR method used in our study was found to be below 15%, and it was decided to be suitable for routine laboratory studies.