title: Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of plasmodium by amplifying RNA and DNA of the 18S rRNA genes.
abstract: A highly sensitive genus-specific quantitative reverse transcriptase real-time PCR (qRT-PCR) assay for detection of Plasmodium has been developed. The assay amplifies total nucleic acids (RNA and DNA) of the 18S rRNA genes with a limit of detection of 0.002 parasite/μl using cultured synchronized ring stage 3D7 parasites. Parasite densities as low as 0.000362 parasite/μl were detected when analyzing clinical samples. Analysis of clinical samples showed that detection of 18S rRNA genes from total nucleic acids increased the analytical sensitivity of the assay by more than 1 log unit compared to DNA only. When clinical samples with no parasites present by microscopy were analyzed by qRT-PCR, 90% (117 of 130) were positive for the presence of Plasmodium nucleic acids. Quantification of clinical samples by qRT-PCR using total nucleic acid versus DNA was compared to microscopy. There was a significantly greater correlation of parasite density to microscopy when DNA alone was used than with total nucleic acid. We conclude that analysis of total nucleic acids by qRT-PCR is a suitable assay for detection of low parasite levels in patients with early-stage malaria and/or submicroscopic infections and could greatly benefit malaria diagnosis, intervention trials, and malaria control and elimination efforts.
authors.affiliation: Division of Malaria Vaccine Development, Center for Molecular Diagnostics and Genomic Studies, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD, USA. Edwin.kamau@us.army.mil
authors.forename: Edwin
authors.initials: E
authors.lastname: Kamau
authors.forename: Ladonna S
authors.initials: LS
authors.lastname: Tolbert
authors.forename: Luke
authors.initials: L
authors.lastname: Kortepeter
authors.forename: Michael
authors.initials: M
authors.lastname: Pratt
authors.forename: Nancy
authors.initials: N
authors.lastname: Nyakoe
authors.forename: Linda
authors.initials: L
authors.lastname: Muringo
authors.forename: Bernard
authors.initials: B
authors.lastname: Ogutu
authors.forename: John N
authors.initials: JN
authors.lastname: Waitumbi
authors.forename: Christian F
authors.initials: CF
authors.lastname: Ockenhouse
mesh_terms: D020459:Genes, rRNA; D006801:Humans; D008288:Malaria / Q000175:diagnosis* / Q000469:parasitology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D010961:Plasmodium / Q000235:genetics / Q000302:isolation & purification*; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D012337:RNA, Ribosomal, 18S