Method Verification of Inhouse Real-time Polymerase Chain Reaction for Detection of Leishmania Species

Abstract

Objective: Leishmaniasis is a vector-borne disease caused by many Leishmania species which can infect both humans and other mammals. Turkey has special epidemiologic importance in terms of this disease due to being located at the junction of Asia and Europe and containing seven geographical regions with environmental and ecologic differences. Microscopic evaluation may miss diagnosis in cases with low levels of parasitemia. The culture method is not chosen by many laboratories due to being laborious preparation of media and results taking a long time. Molecular methods may assist in diagnosis especially with low parasitemia levels and provide results in a short time, so they have begun to be chosen more often currently. This study aimed to verify the method for inhouse real-time PCR using primers for ITS-1 gene region for routine molecular diagnosis of leishmaniasis. Methods: After parasite counting from Leishmania infantum strain, which was carried out on Novy-MacNeal-Nicolle (NNN) medium in our laboratory, DNA extraction was performed with a commercial kit (QIAamp DNA Blood Mini kit, Qiagen, Germany) according to the manufacturer s recommendations. The number of copies in the reaction was determined from DNA sample and serial dilutions of 1/10 were prepared. Then, inhouse real-time PCR method was applied using primers targeting the ITS-1 gene region to determine limit of detection and high and low positivity rates. Then accuracy and precision studies were performed with these samples for method verification. Amplification was performed with a Light Cycler 96 (Roche, France) device. Results were evaluated with amplification curve analysis. The variation coefficients were calculated via accuracy and precision studies from the obtained results. Results: The parasite counts of Leishmania infantum strain were determined as 17,000 promastigote/ml. With using 400 µl of this sample (6,800 copies), DNA obtained in 100 µl elution buffer. Using inhouse real-time PCR amplification, the detection limit for 2 µl DNA (136 copies) was determined as 10 -3 dilution (0.136 copies/reaction). The high positivity rate was determined as dilution above 2 log10 of limit of detection s and the low positivity rate was determined as dilution above 1 log10 of limit of detections. Efficiency of the method was measured with a regression curve for CT values. The standard curve obtained according to CT values and equivalent promastigote counts was linear (slope: -4.097). There was a significant correlation coefficient found between mean CT values and Leishmania infantum DNA concentrations (R=0.99). Conclusion: Our study determined the variation coefficient for the inhouse real-time PCR method was below 15%, confirming it is appropriate for use in our laboratory for routine molecular diagnosis of Leishmania spp.