Detection of human T-cell leukemia virus type I (HTLV-I) provirus in an infected cell line and in peripheral mononuclear cells of blood donors by the nested double polymerase chain reaction method: comparison with HTLV-I antibody tests.
Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification and a second amplification with the products of the first amplification and primers interior to the first primers. Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF. Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and blood centers.
Matsumoto
C
C
Japanese Red Cross Central Blood Center, Tokyo.
Mitsunaga
S
S
Oguchi
T
T
Mitomi
Y
Y
Shimada
T
T
Ichikawa
A
A
Watanabe
J
J
Nishioka
K
K
D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D006800:Deltaretrovirus Infections / Q000175:diagnosis*; D015481:HTLV-I Antibodies / Q000032:analysis; D015368:Human T-lymphotropic virus 1 / Q000235:genetics*; D006801:Humans; D007158:Immunologic Techniques; D007963:Leukocytes, Mononuclear / Q000382:microbiology; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics*
D004279:DNA, Viral; D015481:HTLV-I Antibodies; D009841:Oligonucleotides