A Real Time PCR Platform for the Simultaneous Quantification of Total and Extrachromosomal HIV DNA Forms in Blood of HIV-1 Infected Patients

• DOI: 10.1371/journal.pone.0111919
• Publication Date: 2014-11-03T19:09:42Z
• Authors: Anna Casabianca; Chiara Orlandi; Benedetta Canovari; Maddalena Scotti; Marcello Acetoso; Massimo Valentini; Enzo Petrelli; Mauro Magnani
• Keywords: Provirus; Viremia; Extrachromosomal DNA; SYBR Green I

Background The quantitative measurement of various HIV-1 DNA forms including total, unintegrated and integrated provirus play an increasingly important role in infection monitoring treatment-related research. We report the development validation a SYBR Green real time PCR (TotUFsys platform) for simultaneous quantification total extrachromosomal patients. This innovative technique makes it possible to obtain both measurements single run starting from frozen blood employing same primers standard curve. Moreover, due identical amplification efficiency, allows indirect estimation level. To specifically detect 2-LTR qPCR method was also developed. Methodology/Findings Primers used spanning highly conserved region were selected found all clades group M same. A 195 samples patients wide range clinical conditions analyzed with 100% success rate, even suppressed plasma viremia, regardless CD4+ or therapy. No significant correlation observed between two current prognostic markers, while moderate high inverse HIV DNA, strong values DNA. Conclusions/Significance Taken together, results support use as another tool, addition traditional assays, which can be estimate state viral infection, risk disease progression monitor effects ART. TotUFsys platform allowed us final result, expressed copy number per microgram 104 CD4+, 12 within working days.