MUTATION SPECIFIC PCR AND DIRECT SOLID-PHASE SEQUENCING ASSAY FOR THE DETECTION OF HEPATITIS-B VIRUS PRE-C/C MUTANTS IN ANTI-HBE-POSITIVE, CHRONIC HEPATITIS-B

Sequence analysis of the HBV DNA from patients with anti-HBe+, chronic hepatitis B revealed that the lack of HBeAg is mostly due to a single G-->A transition at nucleotide position 1896, resulting in a translational stop codon. A point mutation-specific polymerase chain reaction (msPCR) for the detection of this genetic variant was established. Two serologically defined groups of patients with symptomatic chronic hepatitis B (HBeAg+ n=14, anti-HBe+ n=11) were included in this study. Viral DNA from 43 sera (26 eAg+/17 anti-HBe+) was amplified twice, using two different sets of PCR primers. Each set contained the same - strand primer, but the + strand primers differed at their 3'-end, thus being complementary only to the wild-type or to the mutant DNA. Unspecific amplification was ruled out by choosing high annealing temperatures (66 degrees C) and cloned HBV-DNA as specificity controls. Furthermore, the results were compared to our findings obtained with direct solid-phase sequencing of the amplified viral DNA. Using msPCR, we found the mutation in four of 26 of the eAg(+) sera and in 17 of 77 of our anti-HBe+ samples. Mixed virus populations were identified in 13 of 21 cases. Compared to the sequencing results, msPCR is more sensitive and less time consuming for the detection of the stop codon and thus is suitable as a rapid and specific screening method. (C) 1994 Wiley-Liss, Inc.