Detection of HIV‐1 sequences in children using radioactive and colorimetric polymerase chain reactions

Abstract

The detection of HIV-1 proviral DNA in children born to seropositive mothers was studied using the polymerase chain reaction with either a radioactive electrophoretic method or a noval procedure that employs colorimetric microwell visualization. Peripheral blood mononuclear cell lysates from 18 HIV-1 infected children and 28 uninfected subjects were assayed for a 142 bp fragment of DNA from the gag region of HIV-1 using the primer pair SK145-431. Detection of amplified DNA was carried out by hybridization with a radiolabeled SK102 probe, or with a tagged SK102 probe permitting colorimetric detection. The radioactive detection procedure demonstrated 100% specificity and correlated with the serological results. The assay was more sensitive than the p24 antigen test, but two false negative results were obtained. One was from a sample taken at 2 weeks, an age at which undetectable provirus levels were reported in almost all HIV-1 infected newborns. The second was probably due to a low copy number of proviral DNA, as positive results were obtained in all other (6) samples from this child. Comparative analysis in a limited number of specimens of radioactive and colorimetric detection following PCR revealed 100% specificity and comparable sensitivity with 4 discordant results. The results show that PCR is the best method for early diagnosis of HIV-1 infection in pediatric subjects. The study also demonstrated the value of a colorimetric detection method for PCR products. This colorimetric microwell plate procedure may prove a useful technique in routine diagnosis of HIV-1 infection in children.

Performance of the Amplicor human immunodeficiency virus type 1 PCR and analysis of specimens with false-negative results

“…False-negative PCR results can be due to one or a combination of the following: (i) the presence of inhibitors of Taq polymerase or other components of the reaction (2,11,14,19), (ii) a low number of copies of the starting target sequence (3,14,17,23,31), or (iii) sequence variability in the amplicon primer and probe region (7,14,22,27). Resolution of the causes of false-negative PCR results is not straightforward.…”

“…Ambiguity may arise for specimens from patients who have been infected very recently or whose humoral response is impaired; false-positive serological findings may also occur. However, the most important use for diagnostic PCR of proviral DNA is the investigation of infants born to HIV-infected mothers (8,12,17,20), in whom maternal antibodies may be detected for up to 18 months after birth (12), and therefore seropositivity may not be indicative of HIV infection.…”

“…Several groups have described the performance of the primers used in the Amplicor assay, both in prototype versions of the kit and in the final commercial product (3,5,8,14,17,19,20,23,27,29). Studies with the commercially available version of the assay have evaluated it with panels of specimens whose serologic status is known rather than reporting its prospective use as a diagnostic test (14,19,20,23,27).…”

Decay of Human Immunodeficiency Virus Type 1 Unintegrated DNA Containing Two Long Terminal Repeats in Infected Individuals after 3 to 8 Years of Sustained Control of Viremia

“…The PBMCs were separated from whole blood, and total cellular DNA was extracted by cell lysis as previously described (7). The cell lysates were stored at Ϫ20°C until assayed.…”

Level of Human Immunodeficiency Virus DNA in Peripheral Blood Mononuclear Cells Correlates with Efficacy of Antiretroviral Therapy

“…PBMCs were isolated, counted, and aliquoted from whole blood treated with acid citrate dextrose by density gradient separation on Lympholyte-H medium (Cedarlane Laboratories, Hornby, Canada). Total cellular DNA was obtained from the PBMCs by cell lysis and stored at Ϫ20°C, as previously described (9). HIV DNA was detected in 315 (99.1%) of the 318 samples from HIV-infected individuals and quantified in 284 (89.3%) samples in a range of 50 to 8,900 copies per 10 6 PBMCs.…”