Quantitative detection of hepatitis B virus DNA by real-time nucleic acid sequence-based amplification with molecular beacon detection

We have developed a hepatitis B virus (HBV) DNA detection and quantificatio n system based on amplification with nucleic acid sequence-based amplificat ion (NASBA) technology and real-time detection with molecular beacon techno logy. NASBA is normally applied to amplify single-stranded target RNA, prod ucing RNA amplicons. In this work we show that with modifications like prim er design, sample extraction method, and template denaturation, the NASBA t echnique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal natu re of the method, which allows high-throughput applications for nucleic aci d detection. The homogeneous real-time detection allows a closed-tube forma t of the assay, avoiding any postamplification handling of amplified materi al and therefore minimizing the risk of contamination of subsequent reactio ns. The assay has a detection range of 10(3) to 10(9) HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dyn amic range, and high-throughput applicability, making it a viable alternati ve to those based on other amplification or detection methods.