Sensitive Detection of HBsAg Mutants by a Gap Ligase Chain Reaction Assay

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American Society for Microbiology

RESUMO

Hepatitis B virus (HBV) surface gene variants have been associated with diagnostic escape and immune escape following vaccination. The most common mutation observed in these variants is a glycine-to-arginine substitution at amino acid 145 (G145R). In order to sensitively detect the presence of this mutant in serum, a new molecular detection system was developed; in this new system, a gap ligase chain reaction (gLCR) assay was coupled with electrochemiluminescence detection of reaction products. The gLCR assay could detect approximately 10 copies of mutant DNA and could discriminate low levels of mutant DNA in the presence of excess wild-type DNA. Detection of the G145R mutant in clinical specimens was evaluated by testing 56 suspect serum specimens. The G145R mutation was observed in 18 of 28 HBV-DNA-positive samples. The approximate percentage of mutant present in each specimen was calculated by comparison with a standard curve of an increasing ratio of mutant DNA to wild-type DNA. Most samples contained a very low percentage of mutant virus (approximately 5%), with an observed range of approximately 3 to 74%. The G145R mutation was most frequently observed in specimens producing a diagnostic anomaly or from transplant patients but was also observed in specimens from vaccinated individuals and specimens in which HBsAg diagnostic escape was suspected. Therefore, the gLCR assay is a sensitive and specific method for detection of G145R mutants, which could be modified to include the detection of other HBV mutants.

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