An allele specific locked nucleic acid real time quantitative PCR for detection of HBV rtA 181 V and rtN 236 T mutations

Background: Drug-resistant mutations of hepatitis B virus (HBV) are the major causes for unsuccessful therapy for chronic hepatitis B infection (CHB). Adefovir dipivoxil (ADV) has been widely used in patients who failed to lamivudine (LMV) treatment. Early detection of ADV-resistant mutationsis of great clinical significance. In this study, we established an easy-to-use approach, real time allele specific locked nucleic acid quantitative PCR (RT-AS-LNAqPCR) for early quantification of the rtA181V and rtN236T mutations associated with resistance to ADV and focused on its performance evaluation. Methods: Four recombinant plasmids for rtA181 and rtN236 mutations were constructed. The assay was established and evaluated with standard recombinant plasmids and 102 serum samples from patients who experienced with ADV. Results: The linear range of the assay for the detection of rtA181V and rtN236T was between 1×109 copies/μl and 1×102 copies/μl. Sensitivity of the assay was 10-5 in the wild-type background of 1×107 copies/μl. The detection sensitivity of the assay was 0.03% in the detection of clinical samples. RT-AS-LNA-qPCR had a high concordance with direct sequencing in detecting mutations associated with resistance to ADV. RT-AS-LNA-qPCR was more sensitive than direct sequencing in detecting minor variants which could detect these mutations earlier. Conclusions: RT-AS-LNA-qPCR assay was able to sensitively and specifically detect the rtA181V and rtN236T mutations associated with resistance to ADV. This easy-to-use approach may be a useful tool for monitoring ADV resistance mutations in patients with chronic HBV infection and for optimization of ADV therapy.