Detection of primary YMDD mutations in HBV-related hepatocellular carcinoma using hybridization-fluorescence...

Lamivudine is used for the treatment of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). However, HBV-related HCC patients with mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) motif have no response to lamivudine therapy. The detection of YMDD mutations in HBV-related HCC patients may help guide the treatment of HCC. In this study, a simple, sensitive, reliable and cost-effective hybridization-fluorescence polarization assay for the detection of YMDD mutations in HCC was developed. A pair of general primers within the highly conserved region of the HBV polymerase gene was used in an asymmetric PCR. Three probes specific for the corresponding YMDD mutations labeled with different fluorescent reporters hybridized to their target amplicons, and hybridization was indicated by higher fluorescence polarization. The hybridization-fluorescence polarization assay was capable of detecting YMDD mutations at a limit of detection of 10 copies per reaction, and the assay was able to detect minor populations of viruses with primary YMDD mutations as low as 10%. The rates of primary YMDD mutations and the correlation between YMDD mutations and HBV genotypes in 251 HBV-related HCC patients were investigated using the hybridization-fluorescence polarization assay.