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J Med Primatol. 2014 Feb;43(1):31-43. doi: 10.1111/jmp.12088. Epub 2013 Nov 22.

Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA.

Author information

1
Division of Microbiology, Tulane National Primate Research Center, Tulane University, Covington, LA, USA.

Abstract

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.

KEYWORDS:

AIDS; PCR inhibition; minimum information for publication of quantitative real-time PCR experiments; plasma viral load; quantitative polymerase chain reaction

PMID:
24266615
PMCID:
PMC3891828
DOI:
10.1111/jmp.12088
[Indexed for MEDLINE]
Free PMC Article

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