We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. 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 dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods.Infection with hepatitis B virus (HBV) can cause a spectrum of liver diseases, such as fulminant or chronic hepatitis, cirrhosis, and hepatocellular carcinoma. About 300 million people throughout the world suffer from chronic HBV infection, and 2 billion people have markers indicating past infection with HBV (15,25).HBV is the smallest DNA virus known, and its genome shows a highly compact organization. A unique aspect in the HBV replication cycle is that a pregenomic mRNA serves as a template for the synthesis of the first viral DNA strand by the reverse transcriptase (RT) polymerase of HBV (35). The RNase H activity of the HBV DNA polymerase removes the mRNA during this process, and synthesis of the complementary second DNA strand is then started, generating a partially double-stranded DNA molecule for packaging in virions. When the virus enters the host, this molecule is extended into a fully double-stranded DNA molecule, thus starting a new replication cycle (10,27,28).HBV DNA can be detected in the blood in more than 90% of infected hosts who are positive for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). The use of detection and quantification of HBV DNA has become the preferred method for measuring the quantity of infectious particles and provides important diagnostic and prognostic information, mainly as a marker of virus replication (5,14,20,29,30).Numerous assays are available for detection of HBV DNA, such as the branched DNA (bDNA) assay (6, 13), DNA hybridization assays (9, 24), and quantitative PCR (1,12,22). Some of these assays have only limited sensitivity, however, and detection by PCR may be considered to be laborious and susceptible to contamination. Since HBV DNA loads are highly variable and require a broad dynamic detection range ...

The protein tyrosine kinase p59fyn (Fyn) plays important roles in both lymphocyte Ag receptor signaling and cytokinesis of proB cells. We utilized yeast two-hybrid cloning to identify the product of the tctex-1 gene as a protein that specifically interacts with Fyn, but not with other Src family kinases. Tctex-1 was recently identified as a component of the dynein cytoskeletal motor complex. The capacity of a Tctex-1-glutathione S-transferase fusion protein to effectively bind Fyn from cell lysates confirmed the authenticity of this interaction. Tctex-1 binding required the first 19 amino acids of Fyn and integrity of two lysine residues within this sequence that were previously shown to be important for Fyn interactions with the immunoreceptor tyrosine-based activation motifs (ITAMs) of lymphocyte Ag receptors. Expression of tctex-1 mRNA and protein was observed in all lymphoma lines analyzed, and immunofluorescence confocal microscopy localized the protein to the perinuclear region. Analysis of a T cell hybridoma revealed prominent colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing cytokinesis. Our results provide a unique insight into a mechanism by which Tctex-1 might mediate specific recruitment of Fyn to the dynein complex in lymphocytes, which may be a critical event in mediating the previously defined role of Fyn in cytokinesis.