Hu Y, Zhang W L, Xie S L, Zhao Y, Hu J L, Cai X F, Lai G Q, Huang A L
Key Laboratory of Molecular Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China.
Genet Mol Res. 2012 Jan 9;11(1):53-60. doi: 10.4238/2012.January.9.6.
Early detection of adefovir dipivoxil-resistant mutants during long-term treatment of chronic hepatitis B virus (HBV) infection with this drug is of great clinical importance. We developed an improved reverse dot hybridization test for simple and rapid detection of the rtA181V/T and rtN236T mutations associated with adefovir dipivoxil resistance in chronic hepatitis B patients. Probes were designed for genotypes B, C, and D of this resistance characteristic; a total of 70 clinical samples were analyzed with this improved reverse dot hybridization assay. Its usefulness was validated by comparing with sequencing data. Discordant results were confirmed by subclone sequencing. This reverse dot hybridization assay was sufficiently sensitive to detect 10(3) copies/mL; it also detected adefovir dipivoxil-resistant mutant strains when they comprised more than 5% of a mixed virus population. This reverse dot hybridization array correctly identified adefovir dipivoxil-resistant mutants; it had high concordance (98.5%) with direct sequencing data. There was no clear relationship between the HBV genotype and the development of adefovir dipivoxil-resistant mutants. This reverse dot hybridization assay proved to be simple and rapid for detection of rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil.
在慢性乙型肝炎病毒（HBV）感染患者长期使用阿德福韦酯治疗期间，早期检测阿德福韦酯耐药突变体具有重要的临床意义。我们开发了一种改进的反向点杂交试验，用于简单快速地检测慢性乙型肝炎患者中与阿德福韦酯耐药相关的rtA181V/T和rtN236T突变。针对该耐药特征的B、C和D基因型设计了探针；使用这种改进的反向点杂交试验对总共70份临床样本进行了分析。通过与测序数据比较验证了其有效性。不一致的结果通过亚克隆测序得以确认。这种反向点杂交试验足够灵敏，能够检测到10³拷贝/毫升；当阿德福韦酯耐药突变株在混合病毒群体中占比超过5%时，也能检测到。这种反向点杂交阵列能够正确识别阿德福韦酯耐药突变体；与直接测序数据具有高度一致性（98.5%）。HBV基因型与阿德福韦酯耐药突变体的发生之间没有明确关系。这种反向点杂交试验被证明对于检测与阿德福韦酯耐药相关的rtA181V/T和rtN236T突变简单且快速。
