Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by real-time RT-PCR assay using self-quenched fluorogenic primers.
HIV-1 viral load is a basic marker to evaluate the severity of HIV-1 related diseases and to monitor the effectiveness of treatment. A method based on real-time RT-PCR technology has been developed to quantify HIV-1 RNA using self-quenched fluorogenic primers known as LUX primers. They were used in this study to recognize a low variable gag region of subtype E and B consensus sequences. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with 10 fold serial dilutions of synthetic HIV-gag RNA. A broad range linear relationship (10 to 10(6) copies/ml) was observed between the number of PCR cycles needed to detect a fluorescent signal and the number of RNA copies. The intra- and inter-assay coefficients of variation were 0.72 to 2.54% and 3.14 to 8.83%, respectively, thus indicating good reproducibility. Thirty out of fifty HIV-infected individual plasma samples were quantified by this method and compared with the AMPLICOR HIV-1 Monitor assay, which is widely considered the reference technique for HIV-RNA viral load measurement. The results indicate that the AMPLICOR HIV-1 Monitor assay and real-time RT-PCR using LUX primers are in good agreement (mean difference in log10 copies/ml+/-2 standard deviations = 0.21+/-1.34).
Promso
Somying
S
Virology and Molecular Microbiology Unit, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
Srichunrusami
Chutatip
C
Utid
Kanchala
K
Lulitanond
Veraphong
V
Pairoj
Wantanit
W
Chantratita
Wasun
W
D015658:HIV Infections / Q000097:blood*; D015497:HIV-1; D006801:Humans; D012367:RNA, Viral / Q000097:blood*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013785:Thailand; D019562:Viral Load
D012367:RNA, Viral