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

Year 2019, Volume: 5 Issue: 3, 192 - 198, 31.12.2019

Selma Usluca

https://doi.org/10.19127/mbsjohs.644748

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. 

Keywords

Inhouse realtime PCR, method verification, Leishmania spp.

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