title: Post-extraction stabilization of HIV viral RNA for quantitative molecular tests.
abstract: 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°C for 92 days and in the cDNA format at 45°C for 7 days as determined by real-time PCR. With RNAstable the R(2) 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 R(2) 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.
authors.affiliation: PATH, Seattle, WA 98109, USA. dstevens@path.org
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Stevens
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Crudder
authors.forename: Gonzalo J
authors.initials: GJ
authors.lastname: Domingo
mesh_terms: D018076:DNA, Complementary / Q000235:genetics; D003890:Desiccation; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D020871:RNA Stability; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D019562:Viral Load / Q000379:methods*; D014773:Virology / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral