Detection of the YMDD motif mutation of P gene of hepatitis B virus using polymerase chain reaction-restriction fragment length polymorphism.
OBJECTIVE: To establish a convenient approach to implement polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for detecting of gene mutation. METHODS: Serum samples were collected from l0 patients with chronic hepatitis B who had received oral lamivudine for at least l year, and 3 methods including mismatched PCR-RFLP, direct sequence analysis of the PCR product and cloning prior to secondary sequence analysis were employed respectively to examine the YMDD motif mutation of P gene of hepatitis B virus (HBV). RESULTS: PCR-RFLP revealed wild-type HBV infection in 7 cases mixed infection in 1 case and HBV mutant infection in 2 cases. With direct sequence analysis of the PCR product, 9 cases were found with Wild-type HBV infection and 1 with HBV mutant infection. However, 2 non-wild-type HBVV-infected cases as approved by PCR-RFLP, opposite to the results by direct sequence analysis, were both shown by sequence analysis after cloning to suffer mixed infection. CONCLUSION: Sequence analysis following cloning is the best way to detect YMDD motif mutation in HBV, but in terms of economy and practicability, mismatched PCR-RFLP is of greater significance in mass screening of YMDD motif mutant HBV than the other 2 approaches.
Zhou
Zhi-Yong
ZY
Department of Infectious Diseases, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China.
Sun
Jian
J
Chen
Jin-Jun
JJ
Hou
Jin-Lin
JL
Luo
Kang-Xian
KX