Detection and Quantification of Human Immunodeficiency Virus RNA in Patient Serum by Use of the Polymerase Chain Reaction

Abstract

Human immunodeficiency virus (HIV) RNA was detected and quantified in the serum of HIV-seropositive individuals using the polymerase chain reaction (PCR) and a nonisotopic enzyme-linked affinity assay. Of 55 HIV-infected patients who were not receiving therapy, serum HIV RNA was detected in 9 of 19 who were asymptomatic, 11 of 16 with AIDS-related complex (ARC), and 18 of 20 with AIDS, with copy numbers ranging from 10(2) to greater than or equal to 5 x 10(4) 200 microliters of serum based on a relationship between absorbance and known copy number of gag gene RNA. Linear regression analysis demonstrated a correlation between infectious titer in 42 patient sera cocultured with donor peripheral blood mononuclear cells (PBMC) and PCR product absorbance (r = .70, P less than .01). Serum HIV RNA detected by PCR also correlated with serum p24 antigen positivity, CD4 counts less than 400/mm3, and the presence of HIV-related symptoms or disease. Quantification of infectious HIV RNA in cell-free serum by PCR may be useful as a marker for for disease progression or in monitoring antiviral therapy.

Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis

“…Although easy to perform, a mere quantification of amplification products after PCR may lead to erroneous estimations of the target sequence number, especially when the nonexponential phase of PCR is reached or when reaction inhibitors are present in samples. Thus, relatively elaborate PCR protocols have to be followed to keep the amplification efficiency of individual reactions constant (Abbott et al, 1988;Bettens et al, 1991;Ferre et al, 1992;Holodniy et al, 1991 ;Landgraf et al, 1991 ;Oka et al, 1990). Effects of varying amplification efficiency in PCR can be elegantly overcome by competitive PCR (Wang et al, 1989).…”

High-capacity in vitro assessment of anti-hepatitis B virus compound selectivity by a virion-specific polymerase chain reaction assay

“…This results in a 300-fold range of detection, thus permitting the quantitative detection of greater than 100-fold reductions in HBV content while allowing 50% inhibitions to be measured in the most linear, central section of the standard curve. The successful use of biotinylated PCR primers for quantitative HRP-based detection of PCR products was first reported by Holodniy et al (6). Rather than using streptavidin-coated beads, we developed our detection method with streptavidin-coated microtiter wells to allow for a higher capacity and the convenience associated with semiautomation.…”

“…In order to exploit the high-throughput capability offered by 96-well PCR machines, we explored the use of antibody capture (9, 10) in a microtiter format to isolate HBV from supernatants prior to high-capacity PCR. Furthermore, the emergence of enzyme-linked immunosorbent assay-like techniques for quantitation of DNA (6,14,25) makes possible the quantitation of PCR products in the microtiter format.Here we describe a unique integration of microtiter-based systems for the simultaneous in vitro assessment of both the …”

Quantitation of Human Immunodeficiency Virus Provirus and Circulating Virus: Relationship with Immunologic Parameters

“…During the progression of HIV infection, the mean concentration of provirus per milliliter of blood increases by only a factor of three for subjects with the lowest concentrations of CD4+ lymphocytes. Greater variations are observed among individ- [3,4,6,19,22]. For example, in one large study, the percentage of plasma coculture positivity varied from 23% in asymptomatic participants to 82% in patients with AIDS [3].…”