title: Optimization of competitively differentiated polymerase chain reaction in detection of HBV basal core promoter mutation.
abstract: OBJECTIVE
  To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation.
  
  
  METHODS
  Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control. To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CD-PCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection.
  
  
  RESULTS
  The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to 10 micromol/L and mutual primer to about 100 nmol/L. Optimized CD-PCR could detect both mutant and wild strain independent of the amount of templates and the number of PCR cycles. Its detection limit was 10(3) copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg.
  
  
  CONCLUSIONS
  Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.
authors.affiliation: Department of Infectious Diseases, the Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, Guangdong Province, China. xiaomoupeng@hotmail.com
authors.forename: Xiao-Mou
authors.initials: XM
authors.lastname: Peng
authors.forename: Lin
authors.initials: L
authors.lastname: Gu
authors.forename: Xue-Juan
authors.initials: XJ
authors.lastname: Chen
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Li
authors.forename: Yang-Su
authors.initials: YS
authors.lastname: Huang
authors.forename: Zhi-Liang
authors.initials: ZL
authors.lastname: Gao
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics; D006512:Hepatitis B Core Antigens / Q000235:genetics*; D006514:Hepatitis B Surface Antigens / Q000097:blood; D006513:Hepatitis B e Antigens / Q000097:blood; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic
chemical_list: D017931:DNA Primers; D004274:DNA, Recombinant; D004279:DNA, Viral; D006512:Hepatitis B Core Antigens; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens