title: Plasmodium chabaudi: reverse transcription PCR for the detection and quantification of transmission stage malaria parasites.
abstract: We have developed two reverse transcription polymerase chain reaction (RT-PCR) techniques to detect and quantify the transmission stages (gametocytes) of Plasmodium chabaudi malaria parasites. Both the qualitative and quantitative techniques are based on the amplification of mRNA coding for the P. chabaudi protein Pcs230, which is expressed exclusively in gametocytes. The quantitative RT-PCR (qRT-PCR) technique was developed and validated by examining serial dilutions of known gametocyte densities. The method generated a high correlation between calibration curves of blind samples (R(2)=0.86). The technique was found to be specific, reproducible, and time efficient for quantification of both patent and sub-patent gametocytemia with a sensitivity level 100-1000 times greater than microscopy. The qualitative RT-PCR (RT-PCR) technique was used to monitor the persistence and dynamics of P. chabaudi gametocytes following acute infection. Mice in two independent experiments were sampled for up to 87 days post-infection. RT-PCR showed that gametocytes can persist for up to 8 weeks, post-infection, whereas microscopy could only detect gametocytes up to 6 weeks. Potential applications of the above techniques for studying the ecology, evolution, and epidemiology of malaria transmission are discussed.
authors.affiliation: Institutes of Evolution, Immunology and Infection Research, Ashworth Laboratories, School of Biological Science, University of Edinburgh, The Kings Buildings, West Mains Road, Edinburgh EH9 3JT, UK. a.r.wargo@ed.ac.uk
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Wargo
authors.forename: Nadine
authors.initials: N
authors.lastname: Randle
authors.forename: Brian H K
authors.initials: BH
authors.lastname: Chan
authors.forename: Joanne
authors.initials: J
authors.lastname: Thompson
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Read
authors.forename: Hamza A
authors.initials: HA
authors.lastname: Babiker
mesh_terms: D000818:Animals; D016054:DNA, Protozoan / Q000097:blood / Q000302:isolation & purification; D005260:Female; D008288:Malaria / Q000469:parasitology* / Q000635:transmission; D051379:Mice; D008815:Mice, Inbred Strains; D016788:Plasmodium chabaudi / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D015800:Protozoan Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000097:blood / Q000302:isolation & purification; D016053:RNA, Protozoan / Q000097:blood / Q000302:isolation & purification*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan