Rapid, sensitive, specific, and quantitative detection of human T-cell leukemia virus type 1 sequence in peripheral blood mononuclear cells by an improved polymerase chain reaction method with nested primers.
Improving on the nested double polymerase chain reaction (PCR) described previously, we have developed a new two-step PCR (TS-PCR) method for detecting more specifically the human T-cell leukemia virus type 1 (HTLV-1) proviral sequences in peripheral blood mononuclear cells (PBMC). In our TS-PCR method, the point of modification is to use optimal concentrations of primers in the first amplification step in the range of 0.01-0.025 microM. This increases sensitivity and specificity enough to detect from 1 to 10(5) copies of template DNA without radioisotopes. This method is rapid because of completion in 1 day and is also applicable for quantitative detection of clinical specimens. The data show that the quantitative detection of HTLV-1 proviral sequences by this method correlates with the anti-HTLV-1 antibody titers from serologic analysis of seropositive healthy carriers. Moreover, the TS-PCR method using each specific primer was also attempted for successful detection of other viral genomes; therefore, the principle of this method is widely suitable for routine detection of genomes in the basic and clinical microbiological fields.
Aono
Y
Y
Shionogi Institute for Medical Science, Osaka, Japan.
Imai
J
J
Tominaga
K
K
Orita
S
S
Sato
A
A
Igarashi
H
H
D001483:Base Sequence; D002460:Cell Line; D015342:DNA Probes; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004279:DNA, Viral / Q000302:isolation & purification; D015481:HTLV-I Antibodies / Q000032:analysis; D015368:Human T-lymphotropic virus 1 / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000382:microbiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D015481:HTLV-I Antibodies