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

被引：26
作者：
Monjure, C. J. [1 ]
Tatum, C. D. [1 ]
Panganiban, A. T. [1 ,2 ]
Arainga, M. [3 ]
Traina-Dorge, V. [1 ,2 ]
Marx, P. A., Jr. [1 ,4 ]
Didier, E. S. [1 ,4 ]
机构：
[1] Tulane Univ, Tulane Natl Primate Res Ctr, Div Microbiol, Covington, LA USA
[2] Tulane Univ, Sch Med, Dept Microbiol & Immunol, New Orleans, LA 70112 USA
[3] Tulane Univ, Div Immunol, Tulane Natl Primate Res Ctr, Covington, LA USA
[4] Tulane Univ, Sch Publ Hlth & Trop Med, Dept Trop Med, New Orleans, LA USA
来源：
JOURNAL OF MEDICAL PRIMATOLOGY | 2014年 / 43卷 / 01期
关键词：
AIDS; minimum information for publication of quantitative real-time PCR experiments; PCR inhibition; plasma viral load; quantitative polymerase chain reaction; REVERSE TRANSCRIPTION-PCR; POLYMERASE CHAIN-REACTION; REAL-TIME PCR; INTERNAL CONTROL; VIRAL LOAD; BRANCHED DNA; ASSAY; SIV; QUANTITATION; VALIDATION;
D O I：
10.1111/jmp.12088
中图分类号：
S85 [动物医学（兽医学）];
学科分类号：
0906 ;
摘要：
IntroductionQuantification 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. MethodsThe 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. ResultsModifications 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. ConclusionsQuantification 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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收藏
页码：31 / 43
页数：13
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