phosphorus-radioisotopes and Hepatitis-B

A rapid assay for the quantification of hepatitis B virus DNA in human serum was developed. The principle of the method combines competitive polymerase chain reaction (cPCR) (for the controlled amplification of hepatitis B virus DNA) and scintillation proximity assay (SPA) technology (for rapid detection and quantitation of PCR products). It also incorporates a reproducible and simple method for the preparation of serum DNA suitable for PCR amplification. The assay has a better linear dynamic range than traditional methods that use 32P to detect PCR products. It was applied to a range of hepatitis B virus (HBV) surface antigen positive (HBsAg + ) sera, and shown to be more sensitive than a commercially available HBV DNA kit.

Topics: Diagnostic Techniques, Radioisotope; DNA, Viral; Gene Amplification; Hepatitis B; Hepatitis B virus; Humans; Phosphorus Radioisotopes; Polymerase Chain Reaction; Reagent Kits, Diagnostic; Reproducibility of Results; Scintillation Counting; Sensitivity and Specificity; Viral Load

A nonradioactive hybridization assay for the detection of hepatitis B virus (HBV) DNA in serum with a digoxigenin-labeled probe is described. The probe was sensitive, being able to detect 0.25 pg of homologous HBV DNA, equivalent to 7 x 10(4) genome copies. After extraction of DNA from clinical samples, the probe detected HBV DNA in 11 of 12 hepatitis B e antigen-positive sera and did not react with 6 hepatitis B surface antigen-negative sera. This result was comparable to that obtained with a radiolabeled probe. When serum samples were treated by the alkaline denaturation method, some false-positive reactions were apparent with the digoxigenin-labeled probe, although their frequency could be reduced to around 8% by modifying the sample treatment with a centrifugation step. Overall, the sensitivity and specificity of the digoxigenin-labeled probe indicate that it is a viable alternative to the radiolabeled probe for the detection of HBV DNA in serum. The lack of radioactive reagents in the digoxigenin labeling and detection system and its long shelf-life make this system suitable for routine use in laboratories.

Topics: Digoxigenin; DNA Probes; DNA, Viral; Evaluation Studies as Topic; Hepatitis B; Hepatitis B virus; Humans; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Sensitivity and Specificity; Virus Replication

Serum HBV-DNA is considered the best parameter for monitoring HBV replication in the liver. The filter hybridization assay (spot test) with 32P-labelled HBV-DNA has been the technique more frequently used to date. A simple solution hybridization assay, in which 125I HBV-DNA is used as labelled probe, has been recently standardized. We have compared the performances of these two assays for the detection of HBV-DNA. The results were similar with the two methods: an agreement was found in 39/44 (89%) samples. Three sera were positive only by the spot assay-and two only by the liquid phase assay. However, in these cases, HBV-DNA levels were near the sensitivity limits of the assay. Therefore, the filter and the liquid phase assays can be considered to be suitable methods to monitor HBV replication, a fundamental index for the clinical assessment and prognosis of patients with HBsAg positive chronic hepatitis.

Topics: Cloning, Molecular; DNA, Viral; Follow-Up Studies; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Radioisotope Dilution Technique