反向斑点杂交法检测HBV基本核心启动子区A1762T/G1764A突变的实验研究

Detection of A1762T/G1764A mutations in the basic core promoter of hepatitis B virus by reverse dot blot hybridization

目的建立一种基于尼龙膜的反向斑点杂交法,用于检测乙型肝炎病毒(HBV)基本核心启动子区(BCP)A1762T/G1764A突变。方法根据我国HBV主要流行的基因型为B和C,从GenBank上查出4种HBV BCP序列。利用在线工具Clustal W进行比对,针对该突变位点设计引物和检测探针。探针经合成和修饰后点在带正电的尼龙膜上。将反向斑点杂交法结合地高辛检测试剂盒用于检测A1762T/G1764A突变,以测序法确定该区域序列的标本为检测对象。结果反向斑点杂交法分别检测5例A1762/G1764病毒株、2例T1762/G1764病毒株、5例A1762/A1764病毒株和4例T1762/A1764病毒株,结果与测序完全相同。结论应用本方法可以快速、准确地HBV相关的热点突变。

Objective To establish a reverse dot blot hybridization based on nylon membrane to detect A1762T/ G1764A mutations in the basic core promoter （BCP） of hepatitis B virus （HBV）. Method The BCP sequences of four HBV strains were collected from GenBank including genotypes B and C. The primers of polymerase chain reaction （PCR） and detec- ting probes were designed after aligning the HBV BCP sequences. The probes were synthesized and modified, which was spot- ted into the nylon membrane. The digoxigenin detection kits were used to display the results of reverse dot blot hybridization. The samples for validation the veracity of the assay were determined by sequencing. Result The results of 5 strains with A1762/G1764, 2 strains with T1762/G1764, 5 strains with A1762/A1764 and 4 strains with T1762/A1764 detected by this method were consistent with those of sequencing. Conclusion The reverse dot blot hybridization method can quickly and ac- curately predict the hot mutations of HBV.