Detection of HIV-1 sequences in children using radioactive and colorimetric polymerase chain reactions.
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.
Giri
A A
AA
Institute of Microbiology, School of Medicine, G. Gaslini Children Hospital, Italy.
Lillo
F B
FB
McDermott
J L
JL
Jannuzzi
C
C
Risso
S
S
Fornia
G L
GL
Concedi
D R
DR
Varnier
O E
OE
D000163:Acquired Immunodeficiency Syndrome / Q000175:diagnosis*; D002648:Child; D002675:Child, Preschool; D003124:Colorimetry; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D015483:HIV Antibodies / Q000097:blood; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D016133:Polymerase Chain Reaction
D004279:DNA, Viral; D015483:HIV Antibodies