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Detection by ligase chain reaction of precore mutant of hepatitis B virus

Authors


Abstract

The clinical diagnosis of infection with the most common precore mutant of hepatitis B virus (HBV), that with a point mutation from guanine to adenine at nucleotide 83 in the precore region, is important because the disease may progress rapidly despite interferon therapy. A practical method to detect this mutant is needed. With the ligase chain reaction (LCR), target DNA sequences can be amplified and single base mutations can be detected. We tried to detect mutant HBV by the LCR alone, but the limit of detection (109 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 few as 102 copies per tube could be detected. The results corresponded with the results of nucleotide sequencing for 22 of the 23 patients. The ratio of clones of mutant and total viruses was estimated for each individual by PCR-coupled LCR. Seroconversion could be identified earlier 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 liver 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 HBV coexisted with mutant HBV in 6 of 7 patients with fulminant hepatitis (FH), and the mean ratio of mutant to total HBV was significantly higher than that in AH. PCR-coupled LCR could be used to detect mutant HBV and to estimate the ratio of mutant to total viruses.

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