[Detection of HBV resistant mutations related to lamivudine, adefovir and entecavir by reverse hybridization technique].
OBJECTIVE
To establish a method for simultaneous detection of HBV resistant mutations associated with three kinds of nucleoside analogues.


METHODS
According to 981 HBV complete sequences in GenBank, two pairs of conserved primers labeled with digoxigenin were synthesized to amplify the region of HBV reverse transcriptase. To detect non-synonymous amino acid substitutions associated with lamivudine, adefovir and entecavir, 26 specific oligonucleotide probes covering ten different codon positions, I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T and M250V/I/L were synthesized and immobilized on nylon membranes charged positively. The oligonucleotide probes immobilized on nylon membranes were then hybridized with PCR products labeled with digoxigenin to detect three drug-resistant mutations. In order to observe specificity and accuracy of probes, HBV wild-type, resistant reference strains and patients serums were assayed by reverse hybridization technique, respectively.


RESULTS
The specific probes of 10 codon positions related to HBV wild-type and resistant reference strains, including I169T, V173L, L180M, A181T, T184G, S202I, M204V, Q215S, N236T, M250V, were distinguished effectively by reverse hybridization method. The results results of 37 samples applicated the method were in accordance with that Of DNA sequencing.


CONCLUSIONS
Reverse hybridization technique can be applied to detect HBV resistant mutations associated with Lamivudine, Adefovir and Entecavir rapidly and accurately.
Liu
Yan-Chen
YC
Institute for Viral Hepatitis, Key Laboratory of Molecular Biology of Infectious Diseases of the Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
Zhang
Wen-Lu
WL
Hu
Yuan
Y
Zhao
Li
L
Lai
Guo-Qi
GQ
Hu
Jie-Li
JL
Yang
Feng
F
Huang
Ai-Long
AL
D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000494:pharmacology; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics*; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000821:virology*; D006801:Humans; D009154:Mutation; D009693:Nucleic Acid Hybridization / Q000379:methods
D000998:Antiviral Agents; D004279:DNA, Viral