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Application of PCR‐LDR‐nucleic acid detection strip in detection of YMDD mutation in hepatitis B patients treated with lamivudine
Author(s) -
Xu Gaolian,
You Qimin,
Pickerill Sam,
Zhong Huayan,
Wang Hongying,
Shi Jian,
Luo Ying,
You Paul,
Kong Huimin,
Lu Fengmin,
Hu Lin
Publication year - 2010
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.21811
Subject(s) - lamivudine , orthohepadnavirus , virology , hepatitis b virus , hepadnaviridae , restriction fragment length polymorphism , polymerase chain reaction , biology , population , locked nucleic acid , microbiology and biotechnology , hepatitis c virus , nucleic acid , virus , dna , medicine , gene , genetics , oligonucleotide , environmental health
Chronic hepatitis B virus (CHBV) infection causes cirrhosis and hepatocellular carcinoma. Lamivudine (LAM) has been successfully used to treat CHBV infections but prolonged use leads to the emergence of drug‐resistant variants. This is primarily linked to a mutation in the tyrosine–methionine–aspartate–aspartate (YMDD) motif of the HBV polymerase gene at position 204. Rapid diagnosis of drug‐resistant HBV is necessary for a prompt treatment response. Common diagnostic methods such as sequencing and restriction fragment length polymorphism (RFLP) analysis lack sensitivity and require significant processing. The aim of this study was to demonstrate the usefulness of a novel diagnostic method that combines polymerase chain reaction (PCR), ligase detection reaction (LDR) and a nucleic acid detection strip (NADS) in detecting site‐specific mutations related to HBV LAM resistance. We compared this method (PLNA) to direct sequencing and RFLP analysis in 50 clinical samples from HBV infected patients. There was 90% concordance between all three results. PLNA detected more samples containing mutant variants than both sequencing and RFLP analysis and was more sensitive in detecting mixed variant populations. Plasmid standards indicated that the sensitivity of PLNA is at or below 3,000 copies per ml and that it can detect a minor variant at 5% of the total viral population. This warrants its further development and suggests that the PLNA method could be a useful tool in detecting LAM resistance. J. Med. Virol. 82: 1143–1149, 2010. © 2010 Wiley‐Liss, Inc.