Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment.
OBJECTIVE
To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionine-aspartate-aspartate (YMDD) motif of HBV to tyrosine-valine-aspartate-aspartate (YVDD) or tyrosine-isoleucine-aspartate-aspartate (YIDD).


METHODS
Cloned wild-type and mutant HBV sequences were used as templates to test the sensitivity and specificity of the assay. A variety of primer construction, primer concentration, dNTP concentration, and annealing temperature of primers were systematically examined. Pair primers specific to rtL180M and rtM204V were selected for YVDD detection. Primer specific to rtM204I with an additional 3'-penultimate base mismatched to both the mutant and wild-type sequence was selected for YIDD detection. We applied this assay to study YMDD mutants in 28 chronic hepatitis B patients before and after lamivudine treatment.


RESULTS
We could detect as little as 0.001%-0.00001% of mutant viruses coexisting in 10(8)-10(9) copies of wild-type HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAg-negative patients before lamivudine treatment. After treatment, two more patients in HBeAg-positive patients and seven more patients in HBeAg-negative patients developed YMDD mutations.


CONCLUSIONS
We developed a highly sensitive and specific assay for detecting YMDD mutants. This assay can be applied to monitor chronic hepatitis B patients before and during lamivudine treatment.
Lee
Cha-Ze
CZ
Departments of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 10016, Taiwan, China. czlee@ha.mc.ntu.edu.tw
Lee
Hsuan-Shu
HS
Huang
Guan-Tarn
GT
Yang
Pei-Ming
PM
Sheu
Jin-Chuan
JC
D020816:Amino Acid Motifs; D000998:Antiviral Agents / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D005814:Genes, Viral; D006509:Hepatitis B / Q000188:drug therapy* / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology*; D009154:Mutation; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics
D000998:Antiviral Agents; D017931:DNA Primers; D018894:Reverse Transcriptase Inhibitors; D014764:Viral Proteins; D019259:Lamivudine