Comparison of Different 18S rRNA Primers with Conventional PCR Methods in Determination of Plasmodium spp. and Evaluation of Nested PCR Method

Abstract

Purpose: In this study, our objective was to develop a conventional PCR method in our laboratory to support microscopy and rapid diagnostic tests in routine diagnosis of malaria. For this purpose, by comparing two different primer sets, it was aimed to determine which primer set gave better results in diagnosis and to use this primer set in future studies.Methods: Microscopic examination is the gold standard for diagnosis of Plasmodium infections. The sensitivity and specificity of conventional PCR method, which was based on two different primer sets with a common gene region, were calculated. Afterward, nested PCR were performed for species differentiation using PCR products obtained with both primer pairs. Results: 165 of 168 blood samples (98.21%), which were microscopically Plasmodium vivax positive, were also positive with rPLU1, rPLU5 primers. Furthermore, 163 of these samples (97.02%) were also positive with rPLU5, rPLU6 primers.In addition, to evaluate whether the method detected all species, PCR was carried out with all species positive samples for both primer pairs. Comparison with microscopic examination showed that sensitivity and specificity of rPLU1 and rPLU5 primer pairs were 98.21% and 100%, respectively while sensitivity and specificity of rPLU5 and rPLU6 primer pairs were 97.02% and 100%, respectively. We found a perfect consistency between microscopy and PCR results with both primer sets. Conclusion:Although there was no significant difference between two primer pairs, which provided better results for cases required a conventional first step PCR method during routine laboratory practice, we decided to prefer rPLU1 and rPLU5 primer pair.