Rapid Detection of Lamivudine-Resistant Hepatitis B Virus Mutations by PCR-Based Methods

Rapid Detection of Lamivudine-Resistant Hepatitis B Virus Mutations by PCR-Based Methods

THAWEESAK CHIEOCHANSIN, SALIN CHUTINIMITKUL, SUNCHAI PAYUNGPORN, APIRADEE THEAMBOONLERS, PISIT TANGKIJVANICH,¹ PIYAWAT KOMOLMIT² and YONG POOVORAWAN

Department of Pediatrics, ¹Department of Biochemistry, and ²Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

Hepatitis B virus (HBV) is one of the major causes of liver disease worldwide, and chronic HBV infection may progress to cirrhosis and hepatocellular carcinoma. Mutations at the active site of DNA polymerase of HBV, tyrosine-methionine-aspartate-aspartate (YMDD) motif, render infected patients resistant to antiviral drug (Lamivudine) therapy. Hence, sensitive and specific methods aimed at detecting the mutants are essential. The purpose of this study was to develop methods for detecting the mutations at YMDD by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real-time PCR using locked nucleic acid (LNA)-mediated TaqMan probes. The results obtained by these methods were compared with those examined by conventional direct sequencing on serum samples of 77 patients treated with lamivudine. Our results show that both PCR-RFLP and real-time PCR could detect wild type, YMDD, and its mutants, tyrosine-isoleucine-aspartate-aspartate and tyrosine-valine-aspartate-aspartate. In addition, the mixtures of the wild-type virus and its mutants in the serum sample were detected. Importantly, real-time PCR is less time-consuming, and more sensitive for the detection of mixed populations than PCR-RFLP. The real-time PCR with LNA-mediated TaqMan probes is a sensitive, specific and rapid detection method for mutations at the YMDD motif, which will be essential for monitoring patients undergoing lamivudine antiviral therapy.

keywords —— HBV, YMDD, Real-time PCR, PCR-RFLP, LNA mediated TaqMan probes

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Tohoku J. Exp. Med., 2006, 210, 67-78

Correspondence: Prof. Yong Poovorawan, M.D., Center of Excellence in Viral Hepatitis Research, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.

e-mail: Yong.P@chula.ac.th