Abstract: Objective To sieve a assay of higher specificity and sensitivity on the spontaneous YMDD mutation in hepatitis B virus. Method Plasmids of YMDD,YVDD,YIDD were constructed with molecular methods. The sensitivity and specificity of the three assays （direct sequencing of PCR products,single probe-specific fluorescent PCR and primer-specific fluorescent PCR）were compared by detecting coexisting strains. Result Plasmids of YMDD,YVDD,YIDD which were constructed successfully were identified by restriction enzyme methods and direct sequence analysis. In mixed plasmids,a certain plasmid can be detected by direct sequencing if it covers more than 20% of the total plasmids. YMDD mutant strains only can be tested by single probe-specific fluorescent PCR when the proportion of the total strains is 50/51. The method of primer-specific fluorescent PCR has higher specificity and sensitivity when mutant strains account for more than 1/11 of the total strains. Conclusions This method of primer-specific real-time PCR is fit for the detection of the spontaneous HBV YMDD mutation.

Key words: Hepatitis B virus, YMDD spontaneous mutation, PCR