Optimization of competitively differentiated polymerase chain reaction in detection of HBV basal core promoter mutation.
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.
Peng
Xiao-Mou
XM
Department of Infectious Diseases, the Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, Guangdong Province, China. xiaomoupeng@hotmail.com
Gu
Lin
L
Chen
Xue-Juan
XJ
Li
Jian-Guo
JG
Huang
Yang-Su
YS
Gao
Zhi-Liang
ZL
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
D017931:DNA Primers; D004274:DNA, Recombinant; D004279:DNA, Viral; D006512:Hepatitis B Core Antigens; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens