Enzymatic gene amplification: qualitative and quantitative methods for detecting proviral DNA amplified in vitro.
We evaluated various detection methods to identify amplified human retroviral sequences after Thermus aquaticus-directed polymerase chain reaction (PCR). A combination of hybridization formats and direct incorporation assays provided the most information. This multiphasic approach enabled us to detect specific human T cell leukemia virus type I (HTLV-I)-homologous regions in several HTLV-I-seronegative patients with T cell lymphoma, as well as variants of HTLV-I and human immunodeficiency virus type 1 in patients with prototype disease. In all diagnostic assays designed to detect a particular retrovirus, it was necessary to include a hybridization step, because sequences (endogenous or exogenous) homologous to certain primers were present in most human DNA preparations and yielded discrete products, sometimes of the predicted molecular weight, after amplification. These products could be discriminated by hybridization from amplified prototype proviral sequences. The intensity of the signal generated after hybridization was proportional to input target DNA, an observation making it feasible to quantitatively measure the proviral load in a DNA sample.
Abbott
M A
MA
Department of Medicine, SUNY Health Science Center 13210.
Poiesz
B J
BJ
Byrne
B C
BC
Kwok
S
S
Sninsky
J J
JJ
Ehrlich
G D
GD
D000386:AIDS-Related Complex / Q000382:microbiology; D000163:Acquired Immunodeficiency Syndrome / Q000382:microbiology; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D004279:DNA, Viral / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D015368:Human T-lymphotropic virus 1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000382:microbiology; D007963:Leukocytes, Mononuclear; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000096:biosynthesis / Q000235:genetics; D011237:Predictive Value of Tests; D011533:Proviruses / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D013824:Thermus / Q000201:enzymology
D004279:DNA, Viral; D009841:Oligonucleotides; D004259:DNA-Directed DNA Polymerase