基于分子倒置探针的乙型肝炎病毒耐药基因单碱基突变检测技术的建立

Molecular inversion probe-based single base mutations of hepatitis B virus resistance gene analysis

目的建立一种检测乙型肝炎病毒耐药基因单碱基突变的分子倒置探针（MIP）检测技术。方法方法学建立。收集第三军医大学大坪医院检验科分离的乙型肝炎病毒（HBV）耐药突变YVDD的野生株和突变株DNA。以HBV耐药基因突变YVDD为研究对象，建立针对该基因突变位点的MIP检测技术。分别采用构建的MIP技术和测序技术对1例HBV耐药突变YVDD野生株和1例突变株进行检测，通过比较MIP技术和测序技术的检测结果，明确MIP技术在临床标本检测中的准确性。结果MIP技术用于单碱基突变检测时采用TaqDNA连接酶和AmpligaseDNA连接酶进行热循环单碱基延伸及连接反应可保证检测的特异性。MIP检测技术的最佳探针浓度为1nmol/L。通过对不同浓度靶序列的检测确定MIP技术的检测灵敏度为1nmol/L。临床标本初步验证发现，MIP技术检测结果与测序方法结果一致。结论成功建立了检测HBV耐药基因单碱基突变的MIP技恭o（中华检验医学杂志。2014，37：337-341）

Objective To establish a molecular inversion probe（MIP） method for detection of single base drug-resistance mutation in Hepatitis B virus （HBV） gene. Methods The HBV wild type and YVDD mutant strain were isolated by Daping Hospital of the Third Military Medical University. The MIP was designed and applied to detect the HBV drug-resistance YVDD mutation in one case of wild type and one case of YVDD mutant HBV strain isolated previously. The results of MIP method were compared with that of sequencing to evaluate the detection accuracy. Results Thermal cycling single-base extension and connection reaction performed by Taq DNA Ligase and Ampligase DNA Ligase could ensure the specificity of the detection. The optimum probe concentration of MIP was 1 nmol/L Through detection of the target gene with different DNA concentrations, the detection sensitivity of MIP was determined as l nmol/L The results of MIP were consistent with that of sequencing method in detection of the clinical samples. Conclusion MIP is successfully used to detect single-base drug-resistance mutation in HBV gene. （ Chin J Lab Med,2014,37： 337-341 ）