Quantification of Plasmodium falciparum sporozoites by ribosomal RNA detection.
We used sequences specific to the small subunit ribosomal RNA (SSU rRNA) of the sporogonic stages of Plasmodium falciparum to design a reserve transcriptase-polymerase chain reaction (RT-PCR) assay that can detect 0.1 sporozoites in total RNA purified from potentially infected mosquitoes. We made a synthetic RNA that is amplified in the RT-PCR by the same primers as the parasite SSU rRNA and that serves as an internal control and competitive quantitation standard. We calibrated the assay for quantitation of sporozoites by making a standard curve with RNA from purified and counted sporozoites. The assay accurately measured sporozoite number with a linear range of at least three orders of magnitude in a single reaction. Some application and limitations of the assay are discussed.
Vernick
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Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Keister
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Toure
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Toure
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YT
D000818:Animals; D000852:Anopheles / Q000469:parasitology*; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D007303:Insect Vectors / Q000469:parasitology*; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics / Q000254:growth & development*; D016133:Polymerase Chain Reaction; D016053:RNA, Protozoan / Q000032:analysis* / Q000737:chemistry; D012335:RNA, Ribosomal / Q000032:analysis* / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase; D012044:Regression Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
D017931:DNA Primers; D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D012194:RNA-Directed DNA Polymerase