title: [Quality evaluation and clinical applicability of pyrosequencing assay kit for detecting hepatitis B virus resistance].
abstract: OBJECTIVE
  To evaluate the quality and clinical applicability of pyrosequencing assay kit for detecting hepatitis B virus resistance (HBV DRT).
  
  
  METHODS
  Serial dilutions of the International Standard for HBV DNA were used to test the detection limit of the PCR for HBV DRT. Plasmids containing the either a wild-type (WT) copy or one of 10 mutant (MT) copies of the HBV RT gene were used to prepare a series of samples with various mutation ratios. To construct the linear relationship between the true mutation rate and the detected mutation rate, each sample was repeated at least 10 times. A total of 102 clinical samples were analyzed by Sanger sequencing and retested by the PCR for HBV DRT to determine the concordance of these two methods.
  
  
  RESULTS
  The lower detection limit of the PCR for HBV DRT was 50 IU/ml. Except for the RT236 MT, the correlation between the true mutation rate and the detected mutation rate for the other nine resistance-related mutation sites were excellent, with R² more than 0.98 (P less than 0.001). Among the 102 clinical samples, four were not amplified successfully by PCR. The results were significantly different between the PCR for HBV DRT method and the Sanger sequencing method (x² = 71.2, P less than 0.001), and concordance was observed for 897/969 (92.6%) amino acid positions in 98 samples. Concordant results were achieved in 46/98 (46.9%) samples at all 10 mutation sites. For detection of a single mutation site, concordance rates ranged from 71.5% to 100% at the 10 mutation sites, respectively. Analysis of discordant samples showed that in 87.5% (63/72), Sanger sequencing detected WT and the PCR for HBV DRT detected WT/MT. In 5.6% (4/72) of samples, Sanger sequencing detected WT/MT and the PCR for HBV DRT detected WT. In the remaining 6.9% (5/72) of samples, Sanger sequencing detected WT but PCR for HBV DRT detected MT.
  
  
  CONCLUSIONS
  The PCR for HBV DRT showed high sensitivity and accuracy in detecting antiviral drug-resistant mutations. The method is superior to Sanger sequencing for detecting minor mutations and can be used for early detection of a resistance mutation.
authors.affiliation: Hepatology Unit and Department of Infectious Disease, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Zhidan
authors.initials: Z
authors.lastname: Zheng
authors.forename: Sheng
authors.initials: S
authors.lastname: Shen
authors.forename: Shaofei
authors.initials: S
authors.lastname: Sui
authors.forename: Hua
authors.initials: H
authors.lastname: Chen
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Jian
authors.initials: J
authors.lastname: Sun
mesh_terms: D000998:Antiviral Agents; D001483:Base Sequence; D024882:Drug Resistance, Viral; D006515:Hepatitis B virus; D006801:Humans; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D000998:Antiviral Agents