Abstract: Objective To establish a peptide nucleic acid clamping PCR assay for detecting hepatitis B virus (HBV) drug
resistance mutation. Methods RtM204I (ATT) mutant, rtM204V (GTG) mutant and rtM204 (ATG) wild-type plasmids mixed at
different ratios were detected for mutations by PNA clamping PCR assay and direct sequencing, and the sensitivity and
specificity of the two methods were compared. Serum samples from 85 patients with chronic HBV infection were detected for
drug resistance using the two methods. Results The sensitivity of PNA-PCR assay was 0.001% in a 105-fold excess of wild-type
HBV DNA with a detection limit of 101 copies. The sensitivity of direct sequencing was 10% with a detection limit of 104 copies.
Mutants were detected in 73 of the 85 serum samples (85.9%), including YIDD in 40 samples, YVDD in 23 samples, and YIDD+
YVDD in 10 samples. The agreement of PNA-PCR assay with direct sequencing was only 40% (34/85, YIDD in 21 samples,
YVDD in 11 samples, and YIDD+YVDD in 2 samples). Neither of the two methods yielded positive results for the negative
control samples, suggesting their good specificity. Conclusion PNA-PCR assay appears to be a more sensitive and rapid assay
for detection of HBV genotypic resistance.