Fast and sensitive quantitative detection of HIV DNA in whole blood leucocytes by SYBR green I real-time PCR assay.
The aim of this study was the development of a real-time PCR for HIV DNA quantification in whole blood leucocytes providing an alternative assay to those already described, almost based on the gag gene detection. The technique (pbs-rtPCR assay) is more rapid (the whole assay required less than 5h), easy to perform, omitting both PBMC purification step and data normalization to a housekeeping gene, when compared to previously published assays. Our method is able to detect all group M HIV-1 subtypes in the highly conserved primer-binding site (PBS) region and to avoid the interference by retroviral endogenous sequences in HIV DNA quantification. The sensitivity was 100% for 2 copies even in the presence of high amounts of genomic DNA (1 microg). To monitor the HIV DNA level in all possible clinical conditions, the assay has been validated and compared with a previously developed gag-PCR assay on 73 HIV-1 HAART-treated patients with a plasma HIV-1 RNA range from <50 to >500,000 copies/ml. The 50% of the samples with a viremia below the limit of detection (50 copies/ml) was found to contain HIV DNA between 2 and 10 copies/microg DNA. The pbs-rtPCR offers a significant improvement in the percentage of quantitatively detectable sample (99%) compared with the gag-PCR (42%) suggesting caution in the choice of HIV DNA assay.
Casabianca
Anna
A
Institute of Biological Chemistry "Giorgio Fornaini", University of Urbino, via Saffi 2, 61029 Urbino (PU), Italy. a.casabianca@uniurb.it
Gori
Caterina
C
Orlandi
Chiara
C
Forbici
Federica
F
Federico Perno
Carlo
C
Magnani
Mauro
M
D052160:Benzothiazoles; D001665:Binding Sites; D017931:DNA Primers / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification; D003959:Diamines; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007962:Leukocytes / Q000821:virology*; D009930:Organic Chemicals / Q000032:analysis*; D011804:Quinolines; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D019562:Viral Load
D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I