Post-extraction stabilization of HIV viral RNA for quantitative molecular tests

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• 作者：Daniel S. Stevens ^{dstevens@path.org} ; Christopher H. Crudder ^{ccrudder@hotmail.com} ; Gonzalo J. Domingo ; ^{gdomingo@path.org}
• 关键词：ART ; anti-retroviral therapy ; DBS ; dried blood spots ; DPS ; dried plasma spots ; G2 LTR RT-PCR ; real-time reverse-transcriptase polymerase chain reaction based viral load detecting the long terminal repeat domain of HIV ; LOD ; limit of detection ; RT-PCR ; rea
• 刊名：Journal of Virological Methods
• 出版年：2012
• 期刊代码：180_01660934
• 类别：med
• 出版时间：June, 2012
• 卷：182
• 期：1-2
• 页码：104-110
• 文件大小：451 K

摘要

Two approaches to stabilize viral nucleic acid in processed clinical specimens were evaluated. HIV-1 RNA extracted from clinical specimens was stabilized in a dry matrix in a commercial product (RNAstable, Biomatrica, San Diego, CA, USA) and in a reverse-transcription reaction mixture in liquid form as cDNA. As few as 145 HIV-1 genome copies of viral RNA are reliably stabilized by RNAstable at 45 掳C for 92 days and in the cDNA format at 45 掳C for 7 days as determined by real-time PCR. With RNAstable the R² at days 1, 7, and 92 were 0.888, 0.871, and 0.943 when compared to baseline viral load values. The cDNA generated from the same clinical specimens was highly stable with an R² value of 0.762 when comparing viral load determinations at day 7 to baseline values. In conclusion viral RNA stabilized in a dry RNAstable matrix is highly stable for long periods of time at high temperatures across a substantial dynamic range. Viral RNA signal can also be stabilized in liquid in the form of cDNA for limited periods of time. Methods that reduce reliance on the cold chain and preserve specimen integrity are critical for extending the reach of molecular testing to low-resource settings. Products based on anhydrobiosis, such as the RNAstable should be evaluated further to support viral pathogen diagnosis.