Improved PCR methods for detection of African rabies and rabies-related lyssaviruses.
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.
Coertse
Jessica
J
Department of Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria 0001, South Africa.
Weyer
Jacqueline
J
Nel
Louis H
LH
Markotter
Wanda
W
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
D015345:Oligonucleotide Probes
Crit Rev Microbiol. 2007;33(4):301-24
18033596
Emerg Infect Dis. 2007 Jan;13(1):136-9
17370529
Arch Virol. 2006 Oct;151(10):2021-35
16705370
Virus Res. 2008 Jul;135(1):10-21
18359532
Clin Infect Dis. 2008 Dec 1;47(11):1410-7
18937576
Virus Res. 2005 Jul;111(1):28-43
15896400
J Virol Methods. 2008 Aug;151(2):317-20
18572257
World Health Organ Tech Rep Ser. 2005;931:1-88, back cover
16485446
J Virol Methods. 1996 Mar;57(1):1-14
8919819
Virus Res. 2003 Nov;97(2):65-79
14602198
Vaccine. 2004 May 7;22(15-16):1921-9
15121304
J Clin Microbiol. 2005 Jun;43(6):2786-92
15956398
Emerg Infect Dis. 2003 Dec;9(12):1623-5
14720408
J Clin Microbiol. 1997 Nov;35(11):2762-6
9350729
Virus Res. 2010 May;149(2):197-210
20138934
Clin Chem. 2009 Apr;55(4):611-22
19246619
Onderstepoort J Vet Res. 1998 Dec;65(4):297-303
10192843
Emerg Infect Dis. 2008 Dec;14(12):1887-9
19046512
Expert Rev Mol Diagn. 2010 Mar;10(2):207-18
20214539
J Clin Microbiol. 2004 Jan;42(1):299-306
14715769
Emerg Infect Dis. 2006 Dec;12(12):1913-8
17326944
J Med Virol. 2009 Aug;81(8):1484-97
19551825
J Virol Methods. 2002 Aug;105(1):25-35
12176139
BMC Mol Biol. 2006 Feb 16;7:5
16483373