Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA
Corresponding Author
C.J. Monjure
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Correspondence
Christopher J. Monjure, Division of Microbiology, Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433, USA.
Tel.: 985 871 6292
fax: 985 871 6248
e-mail: [email protected]
Search for more papers by this authorC.D. Tatum
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Search for more papers by this authorA.T. Panganiban
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Department of Microbiology and Immunology, School of Medicine, Tulane University, New Orleans, LA, USA
Search for more papers by this authorM. Arainga
Division of Immunology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Search for more papers by this authorV. Traina-Dorge
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Department of Microbiology and Immunology, School of Medicine, Tulane University, New Orleans, LA, USA
Search for more papers by this authorP.A. Marx Jr
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA, USA
Search for more papers by this authorE.S. Didier
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA, USA
Search for more papers by this authorCorresponding Author
C.J. Monjure
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Correspondence
Christopher J. Monjure, Division of Microbiology, Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433, USA.
Tel.: 985 871 6292
fax: 985 871 6248
e-mail: [email protected]
Search for more papers by this authorC.D. Tatum
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Search for more papers by this authorA.T. Panganiban
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Department of Microbiology and Immunology, School of Medicine, Tulane University, New Orleans, LA, USA
Search for more papers by this authorM. Arainga
Division of Immunology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Search for more papers by this authorV. Traina-Dorge
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Department of Microbiology and Immunology, School of Medicine, Tulane University, New Orleans, LA, USA
Search for more papers by this authorP.A. Marx Jr
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA, USA
Search for more papers by this authorE.S. Didier
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA
Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA, USA
Search for more papers by this authorAbstract
Introduction
Quantification of plasma viral load (PVL) is used to monitor disease progression in SIV-infected macaques. This study was aimed at optimizing of performance characteristics of the quantitative PCR (qPCR) PVL assay.
Methods
The PVL quantification procedure was optimized by inclusion of an exogenous control hepatitis C virus armored RNA (aRNA), a plasma concentration step, extended digestion with proteinase K, and a second RNA elution step. Efficiency of viral RNA (vRNA) extraction was compared using several commercial vRNA extraction kits. Various parameters of qPCR targeting the gag region of SIVmac239, SIVsmE660, and the LTR region of SIVagmSAB were also optimized.
Results
Modifications of the SIV PVL qPCR procedure increased vRNA recovery, reduced inhibition and improved analytical sensitivity. The PVL values determined by this SIV PVL qPCR correlated with quantification results of SIV RNA in the same samples using the ‘industry standard’ method of branched-DNA (bDNA) signal amplification.
Conclusions
Quantification of SIV genomic RNA in plasma of rhesus macaques using this optimized SIV PVL qPCR is equivalent to the bDNA signal amplification method, less costly and more versatile. Use of heterologous aRNA as an internal control is useful for optimizing performance characteristics of PVL qPCRs.
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