title: Improved PCR methods for detection of African rabies and rabies-related lyssaviruses.
abstract: Eleven different lyssavirus species, four of which occur on the African continent, are presently recognized. These viruses cause rabies, the burden of which is highest in the developing world, where routine laboratory diagnosis is often not available. From an epidemiological and control perspective, it is necessary that diagnostic methods detect the diversity of lyssaviruses present in different regions of the world. A published and widely used heminested reverse transcription-PCR (hnRT-PCR) was evaluated for its ability to detect a panel of diverse African lyssaviruses. Due to the limitations experienced for this assay, an alternative hnRT-PCR was developed. The new assay was found to be accurate and sensitive in the detection of African lyssavirus RNA in a variety of clinical specimens. The assay was further adapted to a real-time PCR platform to allow rapid, one-step, quantitative, and single-probe detection, and an internal control for the verification of sample preparation was included. The limit of detection of the real-time PCR assay was 10 RNA copies per reaction, with inter- and intra-assay variability below 4%. Subsequently, in demonstrating utility, both assays were successfully applied to antemortem rabies diagnosis in humans. We believe that the quantitative real-time PCR assay could find application as a routine confirmatory test for rabies diagnosis in the future and that it will serve as a valuable research tool in the biology of African lyssaviruses. Alternatively, the hnRT-PCR assay can be used in laboratories that do not have access to expensive real-time PCR equipment for sensitive diagnosis of lyssaviruses.
authors.affiliation: Department of Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria 0001, South Africa.
authors.forename: Jessica
authors.initials: J
authors.lastname: Coertse
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Weyer
authors.forename: Louis H
authors.initials: LH
authors.lastname: Nel
authors.forename: Wanda
authors.initials: W
authors.lastname: Markotter
mesh_terms: D000349:Africa; D006801:Humans; D018114:Lyssavirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011818:Rabies / Q000175:diagnosis* / Q000821:virology*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods* / Q000592:standards
chemical_list: D015345:Oligonucleotide Probes
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