Plasmodium: a quantitative molecular assay for detection of sporogonic-stage malaria parasites.
We present a molecular assay to detect malaria parasites during sporogonic development in the mosquito host. Specific primers for Plasmodium-specific small-subunit ribosomal RNA sequences not present in mosquito RNA were used in a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. A synthetic RNA quantitative competitor was made which included targets for two primers and a target sequence for a hybridization probe which is also present in the natural parasite ribosome. The heterobifunctional Tth polymerase was used to carry out both reverse transcription and DNA-dependent polymerase chain reaction in a single reaction tube. Ookinetes and sporozoites, the stages from the beginning and end of sporogonic development, respectively, were both recognized in the assay. The assay was calibrated for quantitation of sporozoites by making a standard curve with counted sporozoites. The linear range of the calibrated assay allowed accurate quantitation of parasite number over at least two orders of magnitude, from 10 to 1000 sporozoites, in each RT-PCR reaction.
Laboratory of Parasitic Disease, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
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Vernick
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Barreau
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Seeley
D000330:Aedes / Q000469:parasitology*; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D010964:Plasmodium gallinaceum / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012335:RNA, Ribosomal