Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria

Natália Ketrin Almeida-de-Oliveira Otacílio C Moreira Aline Rosa de Lavigne Leila Mendonça-Lima Guilherme Loureiro Werneck Cláudio Tadeu Daniel-Ribeiro Maria de Fátima Ferreira-da-Cruz About the authors


The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination.


Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy.


The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision.


The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR.


Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.

Key words:
malaria; diagnosis; Plasmodium vivax

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