QUANTIFICATION OF PLASMODIUM-FALCIPARUM SPOROZOITES BY RIBOSOMAL-RNA DETECTION

We used sequences specific to the small subunit ribosomal RNA (SSU rRN A) of the sporogonic stages of Plasmodium falciparum to design a rever se transcriptase-polymerase chain reaction (RT-PCR) assay that can det ect 0.1 sporozoites in total RNA purified from potentially infected mo squitoes. We made a synthetic RNA that is amplified in the RT-PCR by t he same primers as the parasite SSU rRNA and that serves as an interna l control and competitive quantitation standard. We calibrated the ass ay 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 magn itude in a single reaction. Some applications and limitations of the a ssay are discussed.