title: A real time PCR platform for the simultaneous quantification of total and extrachromosomal HIV DNA forms in blood of HIV-1 infected patients.
abstract: BACKGROUND
  The quantitative measurement of various HIV-1 DNA forms including total, unintegrated and integrated provirus play an increasingly important role in HIV-1 infection monitoring and treatment-related research. We report the development and validation of a SYBR Green real time PCR (TotUFsys platform) for the simultaneous quantification of total and extrachromosomal HIV-1 DNA forms in patients. This innovative technique makes it possible to obtain both measurements in a single PCR run starting from frozen blood employing the same primers and standard curve. Moreover, due to identical amplification efficiency, it allows indirect estimation of integrated level. To specifically detect 2-LTR a qPCR method was also developed.
  
  
  RESULTS
  Primers used for total HIV-1 DNA quantification spanning a highly conserved region were selected and found to detect all HIV-1 clades of group M and the unintegrated forms of the same. A total of 195 samples from HIV-1 patients in a wide range of clinical conditions were analyzed with a 100% success rate, even in patients with suppressed plasma viremia, regardless of CD4+ or therapy. No significant correlation was observed between the two current prognostic markers, CD4+ and plasma viremia, while a moderate or high inverse correlation was found between CD4+ and total HIV DNA, with strong values for unintegrated HIV DNA.
  
  
  CONCLUSIONS
  Taken together, the results support the use of HIV DNA as another tool, in addition to traditional assays, which can be used to estimate the state of viral infection, the risk of disease progression and to monitor the effects of ART. The TotUFsys platform allowed us to obtain a final result, expressed as the total and unintegrated HIV DNA copy number per microgram of DNA or 10(4) CD4+, for 12 patients within two working days.
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino (PU), Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Casabianca
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino (PU), Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Orlandi
authors.affiliation: Azienda Ospedaliera Ospedali Riuniti Marche Nord - Presidio Ospedaliero San Salvatore, Pesaro (PU), Italy.
authors.forename: Benedetta
authors.initials: B
authors.lastname: Canovari
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino (PU), Italy.
authors.forename: Maddalena
authors.initials: M
authors.lastname: Scotti
authors.affiliation: Azienda Ospedaliera Ospedali Riuniti Marche Nord - Presidio Ospedaliero San Salvatore, Pesaro (PU), Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Acetoso
authors.affiliation: Azienda Ospedaliera Ospedali Riuniti Marche Nord - Presidio Ospedaliero San Salvatore, Pesaro (PU), Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Valentini
authors.affiliation: Azienda Ospedaliera Ospedali Riuniti Marche Nord - Presidio Ospedaliero San Salvatore, Pesaro (PU), Italy.
authors.forename: Enzo
authors.initials: E
authors.lastname: Petrelli
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino (PU), Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Magnani
mesh_terms: D000328:Adult; D000368:Aged; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics; D005260:Female; D015658:HIV Infections / Q000097:blood* / Q000235:genetics; D015497:HIV-1; D006801:Humans; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004279:DNA, Viral