DETECTION OF HIV-1 RNA BY NUCLEIC-ACID SEQUENCE-BASED AMPLIFICATION COMBINED WITH FLUORESCENCE CORRELATION SPECTROSCOPY

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primar y HIV infection stage. This technique was combined with fluorescence c orrelation spectroscopy (FCS) which enables online detection of the HI V-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA prob e at nanomolar concentration was introduced into the NASBA reaction mi xture and hybridizing to a distinct sequence of the amplified RNA mole cule. The specific hybridization and extension of this probe during am plification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1-1 nM (threshold for unaided FCS detectio n), the number of amplified RNA molecules in the further course of rea ction could be determined. Evaluation of the hybridization/extension k inetics allowed an estimation of the initial HIV-1 RNA concentration t hat was present at the beginning of amplification. The value of initia l HIV-1 RNA number enables discrimination between positive and false-p ositive samples (caused for instance by carryover contamination)-this possibility of discrimination is an essential necessity for all diagno stic methods using amplification systems (PCR as well as NASBA). Quant itation of HIV-1 RNA in plasma by combination of NASBA with FCS may al so be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.