[A study on detection method of lamivudine related mutations in hepatitis B virus polymerase gene].
OBJECTIVE
To establish a simple and accurate method for rapid detection of lamivudine related mutations in hepatitis B virus (HBV) polymerase gene.


METHODS
HBV polymerase gene fragments of covering B and C active region were amplified by nested polymerase chain reaction (nPCR) or nested mismatched PCR. The PCR products were digested with Nde I or Nia III and subjected to electrophoresis on agarose gel, respectively. The patterns of restriction fragment length polymorphism (RFLP) were distinguished. Using this method, thirty patients with chronic hepatitis B and treated with lamivudine for at least one year were analysed for the lamivudine related mutations in polymerase gene. Sixteen cases without lamivudine therapy were used as controls. Some of the patients were also analysed by clone sequencing.


RESULTS
The nested mismatched PCR-RFLP method was simple, accurate and rapid. The whole experiments could be finished in eleven hours. The least titers of HBV DNA which could be detected was 10.3 copies/ml. The wild or mutant strains judged by RFLP were identified by clone sequencing. Mutation in the tyrosine methionine aspartic aspartic acid (YMDD) motif of HBV polymerase gene was found in eight patients and mutations of YMDD motif associated with L526M were found in another three patients. However, there were no such mutations in the control cases.


CONCLUSIONS
The nested PCR-RFLP is considered as a simple and accurate method for rapid detection of lamivudine related mutations in HBV polymerase gene. It is suitable for larger number of sample detection.
Ding
Jing-juan
JJ
Department of Infectious Diseases, Guiyang Medical College, Guiyang 550004, China.
Zhang
Wei-san
WS
Zhang
Li-sha
LS
D000998:Antiviral Agents / Q000494:pharmacology*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D024882:Drug Resistance, Viral / Q000235:genetics; D006515:Hepatitis B virus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology*; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D000998:Antiviral Agents; D019259:Lamivudine; D004259:DNA-Directed DNA Polymerase