[Comparative study on detection of hepatitis B virus mutants in precore region with two methods].
OBJECTIVE
To establish a mismatched polymerase chain reaction restricted fragment length polymorphism (mPCR-RFLP) method for detection of hepatitis B virus (HBV) mutation in precore A1896, and compare with direct sequencing for evaluating its applicability.


METHODS
According to the principle of mPCR, 194bp gene fragments in HBV precore region was amplified. The products of PCR were digested by Bsu36I and subjected to agarose gel electrophoresis. A method for detecting procore A1896 mutation was established by restricted fragment length polymorphism. Totally 134 sera were analyzed by both mPCR-RFLP and direct sequencing methods. Two sera which were identified having mixed infection with precore wild and mutant strains by mPCR-RFLP also were analyzed by cloning and sequencing.


RESULTS
From 134 sera, 117 could be analyzed for HBV precore 1896 situation by mPCR-RFLP method, 109 could be analyzed by sequencing. In 101 sera which could be analyzed by the two methods, 54 were mutant strains and 47 were wild strains. The results of both methods were completely compatible. There was no significant difference in detective rate of HBV precore A1896 mutation between the two methods. The sequences of five clones from one serum which was identified precore mutant by mPCR-RFLP were all A1896 mutant strains. Another serum was identified as mixed infection by mPCR-RFLP, one clone was A1896 mutant strain and four were G1896 wild strains. The results of mPCR-RFLP were verified by cloning.


CONCLUSIONS
Compared with sequencing, the mPCR-RFLP method is simple, accurate and can be used in large-scale surveys and clinical research.
Liu
Yue-hui
YH
Department of Infectious Diseases, Guiyang Medical College, Guizhou 550004, China.
Ding
Jing-juan
JJ
D000328:Adult; D000368:Aged; D004279:DNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D018740:Genetic Heterogeneity; D006509:Hepatitis B / Q000097:blood / Q000821:virology*; D006512:Hepatitis B Core Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D055815:Young Adult
D004279:DNA, Viral; D006512:Hepatitis B Core Antigens