荧光定量聚合酶链反应检测乙型肝炎病毒DNA

Detection of hepatitis B virus by fluorescence quantitative PCR

目的 建立检测HBV病毒DNA的荧光定量PCR法 (FQ PCR) ,并与运用常规凝胶电泳技术观察特异扩增带检测的结果加以比较。方法 合成扩增HBVDNA 314bp特异保守序列的 1对引物及 1条带 2个荧光基团的寡核苷酸探针。用PE 5 70 0型定量PCR仪完成PCR反应及产物的荧光定量检测。同时PCR产物经琼脂糖凝胶电泳 ,EB染色 ,UVP(凝胶成像仪 )检出有 314bp带者为阳性或弱阳性。结果 建立了检测HBVDNA的荧光定量PCR技术 ,用已知HBV阳性模板不同拷贝数的标准溶液测得标准曲线Ct,原始拷贝数在 10 5 ml以上者为阳性 ,用定量及定性PCR 2种方法检测 6 98例血清标本的结果表明 :用FQ PCR技术共检测出 2 0 4例为阳性 ,阳性率 2 9.2 % ;用定性PCR观察到 193例有阳性特异带 ,阳性检出率为 2 7.6 5 %。没有发现用定性PCR检测为阳性而用FQ PCR检测为阴性者。结论 FQ PCR检测HBVDNA较普通定性PCR技术具有操作简便、灵敏度更高 ,减少发生污染可导致假阳性结果的可能性及自动化程度高等优点 。

Objective To set up a Fluorescence quatitative PCR assay(FQ PCR) to detect HBV DNA and to compare the detected results with those from agarose gel electrophoresis/EB staining. Methods A pair of primers and one probe with 2 fluorescent groups were synthesized. The FQ PCR was carried out in Gene Amp5700 Sequence Detection System followed by agarose gel electrophoresis/EB staining detection of the PCR products. Results A quantitative fluorescence PCR assay for detection of HBV DNA was set up. A standard curve was made ploting 3 to 4 concentrations of known positive HBV DNA as template. 698 serum samples were detected by the FQ PCR with positive rate 29.2%(204/698). A 314bp band was identified in 193 samples out of the 698 cases (27.7%). Conclusion The FQ PCR assay for HBV showed high sensitivity, good specificity, more automation without cross contamination and so can be used to detect HBV DNA accurately and quantitatively. [