title: Detection of hepatitis B virus A1762T/G1764A mutant by amplification refractory mutation system.
abstract: OBJECTIVE
  To study the role of hepatitis B virus with A1762T/G1764A double mutation in liver cirrhosis and hepatocellular carcinoma, and create a sensitive, fast, accurate assay for detection of A1762T/G1764A double mutation.
  
  
  METHODS
  We developed an accurate and fast real-time amplification refractory mutation system to detect A1762T/G1764A double mutation. Cloned hepatitis B virus genome was used as a control. Assay sensitivity was determined by serial dilution and mixed template experiments. Specificity was determined by cross experiments with wild and mutant hepatitis B virus. Fifty clinical samples were tested by the real-time amplification refractory mutation system and the results were compared with sequencing.
  
  
  RESULTS
  The real-time amplification refractory mutation system had a sensitivity of 100 copies of virus with these mutations, and 0.1% weak population virus with double mutation could be found in mixtures. A total of 50 randomly collected clinical samples were detected by real-time amplification refractory mutation system, and the results were consistent with those by DNA sequencing. Hepatitis B virus genotype C was more prevalent in 39 of 50 samples than genotype B (11 samples), and about 75% of genotype C carried a double mutation compared to 45% of genotype B. However, the percentage of A1762T/G1764A double mutation in hepatitis B e antigen-negative (58.3%) samples was almost the same as in hepatitis B e antigen-positive (61%) samples.
  
  
  CONCLUSIONS
  The real-time amplification refractory mutation system is sensitive and specific for detection of hepatitis B virus double mutation.
authors.affiliation: Institute for the Study of Liver Diseases, The Third People's Hospital of Changzhou, Changzhou, China.
authors.forename: Yong-Zhong
authors.initials: YZ
authors.lastname: Wang
authors.affiliation: Institute for the Study of Liver Diseases, The Third People's Hospital of Changzhou, Changzhou, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: Institute for the Study of Liver Diseases, The Third People's Hospital of Changzhou, Changzhou, China.
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Zhang
authors.affiliation: Institute for the Study of Liver Diseases, The Third People's Hospital of Changzhou, Changzhou, China.
authors.forename: Min-Zhi
authors.initials: MZ
authors.lastname: Zhu
authors.affiliation: Institute for the Study of Liver Diseases, The Third People's Hospital of Changzhou, Changzhou, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.affiliation: Institute for the Study of Liver Diseases, The Third People's Hospital of Changzhou, Changzhou, China.
authors.forename: Chun-Hua
authors.initials: CH
authors.lastname: Chen
authors.affiliation: Institute for the Study of Liver Diseases, The Third People's Hospital of Changzhou, Changzhou, China. Electronic address: wyz213008@163.com.
authors.forename: Long-Gen
authors.initials: LG
authors.lastname: Liu
mesh_terms: D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000821:virology*; D004279:DNA, Viral / Q000235:genetics*; D005838:Genotype; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008103:Liver Cirrhosis / Q000821:virology*; D008113:Liver Neoplasms / Q000821:virology*; D009154:Mutation / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D004279:DNA, Viral
keywords: Amplification refractory mutation system; Basal core promoter mutations; Hepatitis B virus; PCR