Kd. Vernick et al., PLASMODIUM - A QUANTITATIVE MOLECULAR ASSAY FOR DETECTION OF SPOROGONIC-STAGE MALARIA PARASITES, Experimental parasitology, 81(4), 1995, pp. 436-444
We present a molecular assay to detect malaria parasites during sporog
onic development in the mosquito host. Specific primers for Plasmodium
-specific small-subunit ribosomal RNA sequences nor present in mosquit
o 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 hybridiza
tion 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 beginnin
g and end of sporogonic development, respectively, were both recognize
d in the assay. The assay was calibrated for quantitation of sporozoit
es by making a standard curve with counted sporozoites. The linear ran
ge of the calibrated assay allowed accurate quantitation of parasite n
umber over at least two orders of magnitude, from 10 to 1000 sporozoit
es, in each RT-PCR reaction. (C) 1995 Academic Press, Inc.