A rapid and sensitive method for non-isotopic quantitation of HIV-1 RNA using thermophilic SDA and flow cytometry.
Thermophilic strand displacement amplification (tSDA) is an isothermal DNA amplification technique that proceeds at 55-60 degrees C using both a thermostable restriction enzyme and a DNA polymerase. A modification of this system has been developed that allows the simultaneous amplification and detection of a DNA target by the addition of a detector probe to the reaction. This tSDA system has been further modified into a flow cytometry-based, bead capture assay for quantitation of HIV-1 RNA. A biotinylated capture probe and digoxygenin-dUTP have been incorporated into the tSDA reaction. The resulting double labelled amplicons are captured on strepavidin beads, and a fluorescent signal is generated on the beads by staining with fluorescent anti-digoxygenin antibody. The assay has a linear dynamic range of three orders of magnitude with a lower detection limit at 250 HIV-1 RNA molecules.
Mehrpouyan
M
M
Becton Dickinson Immunocytometry Systems, San Jose, CA 95131-1807, USA.
Bishop
J E
JE
Ostrerova
N
N
Van Cleve
M
M
Lohman
K L
KL
D019921:Biotinylation; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004076:Digoxigenin; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005453:Fluorescence; D005455:Fluorescent Antibody Technique; D015683:Gene Products, gag / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D008863:Microspheres; D021141:Nucleic Acid Amplification Techniques; D010761:Phosphorus Radioisotopes; D018075:RNA, Complementary; D012367:RNA, Viral / Q000032:analysis*; D012044:Regression Analysis; D019809:Streptavidin
D017931:DNA Primers; D015683:Gene Products, gag; D010761:Phosphorus Radioisotopes; D018075:RNA, Complementary; D012367:RNA, Viral; D019809:Streptavidin; C028678:CYCGRG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D004076:Digoxigenin