Comparison of Identification of Toxoplasma gondii by Commercial Realtime PCR and Inhouse Realtime PCR Methods

Year 2019, Volume: 5 Issue: 2, 79 - 84, 28.08.2019

Selma Usluca , Bekir Çelebi

https://doi.org/10.19127/mbsjohs.558436

Abstract

Objective: This study aimed to compare the diagnosis of toxoplasmosis with a
commercial kit and inhouse realtime PCR methods to determine molecular methods
with high diagnostic accuracy for use in addition to serologic tests for
routine diagnosis.

Methods: The study included a total of 116 samples of blood,
CSF or amniotic fluid with 19 identified positive and 97 negatives for T.
gondii sent to our laboratory. Due to the low number of positive samples,
DNA samples from an external quality control program that our laboratory
participates in were included in the study. First to all samples, realtime PCR
method were applied with commercial kit used primers for T. gondii
rep529 gene, and then inhouse realtime PCR were applied with TG-F and TG-R
primers and Taqman probe, targeting the insertion sequence region of T.
gondii B1 gene.

Results: The results for the total of 116 samples studied with both methods was that
17 were identified as positive with commercial realtime PCR and 19 were
determined as positive with inhouse realtime PCR. Accordingly, two cases with
the commercial realtime PCR method were determined as false negative. The limit
of detection for both methods used in our study was determined as 10-3 dilution
(0.028 copy/reaction). There was a high level of compatibility determined
between the inhouse and realtime PCR methods (kappa value: 0.934).

Conclusion: In conclusion, though there was perfect compatibility observed between the
results with the two methods, disadvantages of the commercial realtime PCR
method included isolates where the target gene was not found, deletion or
mutation of all or part of this gene or different numbers of repeats causing
false negative results and high cost. Considering this, our laboratory decided
to use the inhouse realtime PCR using primers for the B1 gene to research T.
gondii with molecular methods. A significant limitation of the study is the
low number of positive samples. For DNA samples belonging to the External
Quality Control Program, the commercial kit was 66.66% successful, while the
inhouse realtime PCR method was 100% successful.

Keywords

Inhouse realtime PCR, realtime PCR, T. gondii

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