Development of Real-Time TaqMan based Assay for the Diagnosis of Malaria

Abstract

Every year Pakistan records 3.5 million suspected and confirmed cases of malaria with P. vivax and P.falciparum infections being the most common. Even though microscopy is the gold standard for malaria diagnosis, it has poor sensitivity and is time consuming. Microscopy has its limits but molecular detection has overcome these problems by providing higher sensitivity and specificity. To reduce disease burden rapid malaria diagnostic procedures that are highly sensitive and specific for early diagnosis along with differentiation of plasmodium species is essential which is critical for treatment of infection A multiplex Real-Time PCR method is designed for the detection and differentiation of Plasmodium into P.malariae, P.vivax, P.falciparum and P.ovale to improve diagnosis and treatment of malaria. One primer set targeting a conserved region of the 18s rRNA gene are designed and this region is variable enough to design four different specie specific TaqMan probes. Real-time PCR detected Plasmodium vivax in 21 samples out of 52 blood samples. The assay has sensitivity of 4.1 parasites/µL for plasmodium vivax. The developed assay is convenient to perform and results are produced in 1 hour and 30 mins. Therefore, this can provide an alternative method for early diagnosis of malaria and can also be used in epidemiological studies.