Rapid and high throughput detection of HBV YMDD mutants with fluorescence polarization.

AIM

To develop a simple and rapid detection of HBV gene variants and prediction of lamivudine-resistance in patients.

METHODS

Initially, plasmids harboring the wild-type or mutant HBV DNA fragments were used in a model system. The technique was then applied to clinical samples for an analysis of YMDD mutations. The sera were extracted from chronic hepatitis patients who had received lamivudine treatment for more than one year. P region gene of HBV was amplified by polymerase chain reaction. The excess primers and dNTPs in PCR products were removed by cleaning-up reagents. Template-directed dye-terminator incorporation reaction was performed and R110 or TAMRA labeled acyclo-terminator was added on the 3' end of TDI-primer specifically. Fluorescence polarization value was measured with Victor 2 multilabel counter and the genotypes of HBV were analyzed.

RESULTS

The YMDD genotypes in recombined positive plasmid and 56 serum samples of HBV infected patients were analyzed by using our TDI-FP method and the specificity and sensitivity were confirmed by DNA sequencing. Five of 56 serum samples showed YVDD phenotype (9%), including 1 YMDD and YVDD mixed infection. Four of 56 showed YIDD phenotype (7.1%).

CONCLUSION

This is a simple, rapid, low cost and high throughput assay to detect HBV polymerase gene variants and suitable for large-scale screening and prediction of the lamivudine-resistance in clinical samples.