PLASMODIUM - A QUANTITATIVE MOLECULAR ASSAY FOR DETECTION OF SPOROGONIC-STAGE MALARIA PARASITES

Citation
Kd. Vernick et al., PLASMODIUM - A QUANTITATIVE MOLECULAR ASSAY FOR DETECTION OF SPOROGONIC-STAGE MALARIA PARASITES, Experimental parasitology, 81(4), 1995, pp. 436-444
Citations number
12
Categorie Soggetti
Parasitiology
Journal title
ISSN journal
00144894
Volume
81
Issue
4
Year of publication
1995
Pages
436 - 444
Database
ISI
SICI code
0014-4894(1995)81:4<436:P-AQMA>2.0.ZU;2-Z
Abstract
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.