Abstract: ObjectiveTo eliminate the opportunity of cross-contamination resulting from the two rounds of preparation of reaction mixtures in the detection of HBV DNA by conventional nested PCR and to improve PCR specificity and sensitivity.MethodsA drop-in,drop-out heminested PCR assay has been developed for the detection of HBV DNA by designing the different length and reaction concentration of primers and different annealing temperatures of initial and secondary PCR amplification.3 Primer s were all present in the initial reaction mix and the nested PCR were completed in a single tube.ResultsThe drop-in,drop-out heminested PCR is capable of amplifying 4 molecules of cloned HBV DNA to levels detectable in ethidium bromid-estained agarose gels.Of 16 specimens which were negative of HBV detected by ELISA and conventional PCR,2 were confirmed HBV DNA positive by drop-in,drop-out heminested PCR.ConclusionThe drop-in,drop-out heminested PCR for HBV achieves the sensitivity of nested PCR in a simplified format that avoids the false-positive results associated with the conventional nested PCR.The positive rate of HBV detection in clinical specimens could be elevated by the combined use of ELISA and drop-in,drop-out heminested PCR,which is of significance in decreasing the HBV infection by transfusion.