Use of Molecular Beacon Probes for Real-Time PCR Detection of Plasmodium falciparum and Other Plasmodium Species in Peripheral Blood Specimens

Abstract

We describe the development and evaluation of a novel pair of real-time, fluorescence-based PCR assays using molecular beacon probes for rapid, sensitive, and specific detection and quantification of Plasmodium falciparum and other Plasmodium species organisms.Diagnosis of human malaria has traditionally rested on microscopic examination of stained peripheral blood films. While relatively inexpensive, this "gold standard" method is laborintensive and time-consuming, requires an experienced microscopist, and may be insufficiently sensitive in detecting low-level parasitemia. Molecular diagnosis of malaria has recently attracted attention as a possible alternative to microscopy. Conventional PCR assays, while demonstrating increased sensitivity and specificity, remain labor-intensive, slow, and prone to potential amplicon contamination problems. The recent emergence of real-time PCR technology addresses these concerns by allowing for rapid, simultaneous amplification, detection, and quantification of target DNA through the use of specific fluorophore-labeled probes or nonspecific DNA-binding dyes. We describe a novel pair of molecular beacon probe-based real-time PCR assays for the sensitive and specific detection and quantification of human malaria parasites, with simultaneous differentiation of Plasmodium falciparum from other members of the genus.Assay development and evaluation were performed at Calgary Laboratory Services, an integrated laboratory serving an urban population of 1.25 million people. Peripheral blood specimens for malaria diagnosis are routinely processed and analyzed according to CLSI standards (1, 15). Positive samples are further retained at Ϫ80°C for conventional species-specific PCR confirmation by a reference laboratory (Toronto General Hospital, Toronto, Canada). In the present study, 28 frozen nonduplicate microscopy-positive peripheral blood specimens and 50 randomly selected microscopy-negative specimens collected over a 25-month period from

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