Simultaneous detection of two major lamivudine-resistant mutants using competitively differentiated-PCR

Rapid, specific and sensitive methods without advanced equipment are required urgently in developing countries in order to detect or monitor lamivudine-resistant mutants routinely before or in the course of the therapy. A protocol is described for the detection of two major YMDD mutations simultaneously through modifying a previous competitively differentiated-PCR (CD-PCR) by revising the strategy, increasing the number of competitively differentiated primers, increasing the number of labeled haptens, optimizing the amplification system and analyzing its products by enzyme immunoassay. Special care was taken to promote the sensitivity, specificity and the ability of the protocol to detect mutation in mixture of the mutants and wild strain. YMDD mutants in clinical serum samples were detected simultaneously, specifically and rapidly only with assistance of the equipment used widely in highly prevalent areas of hepatitis B virus infection.