应用肽核酸反向杂交技术检测HBV阿德福韦耐药突变株

Application of peptide nucleic acid reverse hybridization technology in detecting adefovir-resistant mutations in hepatitis B virus

摘要:

目的：建立一种肽核酸（peptide nucleic acid，PNA） 反向杂交技术，用于检测乙肝病毒阿德福韦酯（adefovir dipivoxil，ADV）常见耐药突变位点。方法：利用反向杂交技术，设计出特异性的PNA探针，与PCR扩增后带有生物素标记的样本进行杂交；通过对探针浓度，杂交温度和时间等条件的优化建立起最佳反应条件。应用该技术检测了临床ADV治疗慢性乙肝患者的72例血清样本，与测序法比较了其检测的准确性。结果：（1）通过共价结合能够将PNA探针固定在尼龙膜上，特异性检测ADV常见耐药突变位点。（2）与直接测序法相比，PNA反向杂交技术检测准确性为97.88％。结论：通过优化杂交反应，建立起快速、灵敏的检测乙肝病毒ADV常见耐药突变位点的PNA反向杂交技术。

Abstract:

Objective：To detect adefovir-resistant mutants of hepatitis B virus（HBV） using peptide nucleic acid（PNA） probes. Methods：Specific PNA probes were designed for genotypes B，C D，which were then hybrized with biotinylated PCR products. Different hy－bridization factors such as probe concentration，hybridization temperature and time were investigated to establish the optimization conditions. Finally，72 hepatitis B virus（HBV） clinical samples with adefovir dipivoxil treatment were investigated using PNA hybriza－tion assay. Its usefulness was validated by comparing with sequencing data. Results：（1）PNA probes could be fixed on the membrane by cross link and used for reverse hybridization. （2）Complete concordance of PNA hybrization assays was 97.88% with direct sequenc－ing. Conclusion：We develop an assay using PNA probes to detect adefovir-resistant mutants of HBV.