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

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 a...

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Bibliographic Details
Published in:	Journal of medical primatology Vol. 43; no. 1; pp. 31 - 43
Main Authors:	Monjure, C.J., Tatum, C.D., Panganiban, A.T., Arainga, M., Traina-Dorge, V., Marx Jr, P.A., Didier, E.S.
Format:	Journal Article
Language:	English
Published:	Denmark Blackwell Publishing Ltd 01-02-2014 Wiley Subscription Services, Inc
Subjects:	AIDS Animals Antiretroviral drugs Drug therapy Genome, Viral Hepatitis C virus HIV Human immunodeficiency virus Macaca mulatta minimum information for publication of quantitative real-time PCR experiments Optimization PCR inhibition Plasma plasma viral load quantitative polymerase chain reaction Real-Time Polymerase Chain Reaction - methods Reproducibility of Results RNA, Viral - blood RNA, Viral - genetics Sensitivity and Specificity Simian Acquired Immunodeficiency Syndrome - blood Simian Acquired Immunodeficiency Syndrome - virology Simian immunodeficiency virus Simian Immunodeficiency Virus - genetics Viral Load quantitative polymerase chain reaction AIDS plasma viral load PCR inhibition minimum information for publication of quantitative real-time PCR experiments
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Summary:	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.
Bibliography:	istex:F55A83E8965B808B24ED5B42BF6F3E2D10B31DD9 ark:/67375/WNG-37VRDW5W-6 ArticleID:JMP12088 Tulane National Primate Research Center NIH - No. OD011104 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Undefined-1 ObjectType-Feature-3 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0047-2565 1600-0684
DOI:	10.1111/jmp.12088