Quantitative analysis of wild-type and HBeAg minus hepatitis B viruses by a sequence-dependent primer extension assay
✍ Scribed by M. R. Brunetto; A. Randone; M. Ranki; A. Jalanko; P. Piantino; M. Giarin; G. Capra; P. L. Calvo; F. Oliveri; Dr. F. Bonino
- Publisher
- John Wiley and Sons
- Year
- 1994
- Tongue
- English
- Weight
- 531 KB
- Volume
- 43
- Category
- Article
- ISSN
- 0146-6615
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✦ Synopsis
Abstract
The ratio between wild‐type hepatitis B virus (HBV) and HBV mutant, unable to secrete “e” antigen (HBeAg minus HBV) appears to be an important determinant of the outcome of chronic hepatitis B. Quantitative analysis of wild‐type and HBeAg minus HBVs in the blood could be useful to monitor chronic hepatitis B patients. We developed a solid‐phase minisequencing assay for both viruses using a primer‐guided incorporation of a single labeled nucleotide on an affinity captured biotinylated amplified HBV‐DNA template. A standard curve was constructed by mixing increasing quantities of wild type and mutant virus DNAs. The detection of wild‐type and HBeAg minus sequences, ranging from 10% to 90% of overall viremia, was linear and reproducible till 0.1 pg/μl of serum HBV‐DNA. The assay yields numerical values and the ratio of incorporated nucleotides defines the relative proportions (%) of the two viral sequences with accuracy. We tested the sensitivity and accuracy of the minisequencing on mixed end point dilutions of wild‐type and HBeAg minus reference sera and amplified products. The feasibility and reproducibility of the assay were tested in 35 sera from 21 HBsAg positive patients with chronic hepatitis B using both minisequencing and oligo‐hybridization assays. A high correlation was found between the two assays (r = 0.957 P < 0.0001). In conclusion, the minisequencing assay provides a precise and reproducible quantitative analysis of wild‐type and HBeAg minus HBVs in clinical specimens. It is proposed to study the relations between HBV heterogeneity and the course of hepatitis B and its response to therapy. © 1994 Wiley‐Liss, Inc.