A rapid real-time PCR assay for the detection of HIV-1 proviral DNA using double-stranded primer.
In this study, a rapid real-time PCR assay to detect HIV-1 proviral DNA in whole blood was developed using a novel double-stranded primer that does not require a target-specific fluorescent probe or intercalating dye systems. Co-amplification of a human gene RNase P served as the internal control to monitor the efficiency of the DNA extraction and PCR amplification. The HIV-1 amplification efficiency was 100% and could amplify 1 copy of HIV-1 DNA 64% of the time and all attempts to amplify 4 copies were successful in less than 51 min. All 22 HIV-1 sero-positive and 20 sero-negative whole blood specimens tested were classified correctly by this assay. In addition, 22 cultured PBMC specimens infected with various HIV-1 subtypes or CRF (A=2, AC=1, B=4, C=3, D=3, AE=2, F=1, BF=2, G=4) were amplified equally well with a similar threshold cycle (C(t)) number (22.9+/-1.2). The high amplification efficiency and short PCR cycles were in part due to the short target sequence amplified by eliminating the probe-binding sequence between the primers. This assay may be useful as an alternative confirmation test in a variety of HIV testing venues.
Pau
Chou-Pong
CP
Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA 30333, United States. chp3@cdc.gov
Wells
Susan K
SK
Rudolph
Donna L
DL
Owen
S Michele
SM
Granade
Timothy C
TC
D001769:Blood / Q000821:virology; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D012015:Reference Standards; D043262:Ribonuclease P / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors
D017931:DNA Primers; D004279:DNA, Viral; D004247:DNA; D043262:Ribonuclease P