DETECTION BY LIGASE CHAIN-REACTION OF PRECORE MUTANT OF HEPATITIS-B VIRUS

The clinical diagnosis of infection with the most common precore mutan t of hepatitis B virus (HBV), that with a point mutation from guanine to adenine at nucleotide 83 in the precore region, is important becaus e the disease may progress rapidly despite interferon therapy. A pract ical method to detect this mutant is needed. With the ligase chain rea ction (LCR), target DNA sequences can be amplified and single base mut ations can be detected, We tried to detect mutant HBV by the LCR alone , but the limit of detection (10(9) copies per tube) was too high. To increase the sensitivity, we used the LCR on DNA already amplified by the polymerase chain reaction (PCR), and tested serum samples from 23 subjects with chronic HBV infection for mutant and wild-type HBV. As f ew as 10(2) copies per tube could be detected. The results corresponde d with the results of nucleotide sequencing for 22 of the 23 patients. The ratio of clones of mutant and total viruses was estimated for eac h individual by PCR-coupled LCR. Seroconversion could be identified ea rlier in the illness by an increase in this ratio than by the decrease in HBeAg. We also tested serum samples from 11 patients with acute li ver failure by PCR-coupled LCR. Mutant HBV was detected at a low ratio in all 4 patients with acute self-limited hepatitis (AH). Wild-type H BV coexisted with mutant HBV in 6 of 7 patients with fulminant hepatit is (FH), and the mean ratio of mutant to total HBV was significantly h igher than that in AH. PCR-coupled LCR could be used to detect mutant HBV and to estimate the ratio of mutant to total viruses.