A rapid chemiluminescence detection method for PCR-amplified HIV-1 DNA.
A solution hybridization assay using acridinium ester labelled probes is described for detection of amplified HIV-1 DNA segments. Amplification was achieved by 30 cycles of the polymerase chain reaction using SK38/SK39 primers specific for a constant region of the HIV-1 gag region together with HLA DQ alpha primers as internal control. Discrimination between hybridized and non-hybridized probes by differential hydrolysis resulted in a three log reduction of the chemiluminescence signal of the non-hybridized probe within 6 min without significant changes in the hybridized probes due to protection of the acridinium ester to hydrolysis by intercalation formation. Chemiluminescence was measured by a two-step-injection method with hydrogen-peroxide and NaOH. About 50 attomols of HIV-1 gag DNA could be detected. Chemiluminescence results, given in relative light units (RLU) of 159 HIV-1-infected patients (range 4013-458319) showed clear discrimination from 64 noninfected control samples (range 838-1477) (cut off 2000 RLU). Comparison with parallel detection of amplified products with autoradiography (32P) and ethidium bromide-stained agarose gels or a p24 antigen ELISA demonstrated better sensitivity and reproducibility of the chemiluminescence assay. The time required for the assay, including measurements, is less than 30 min, which allows reporting 'PCR results' on the same day.
Schmidt
B L
BL
Ludwig Boltzmann Institute for dermato-venerological Serodiagnosis and Dermatological, Hospital City of Vienna, Lainz, Wien, Austria.
D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D015658:HIV Infections / Q000175:diagnosis / Q000382:microbiology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting; D007938:Leukemia; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D004279:DNA, Viral