荧光定量PCR技术在血液筛查中的应用及可行性分析

Application and analysis of feasibility to FQ-PCR in blood screening

目的 了解 ELISA法筛查血液乙型肝炎病毒 ( hepatitis B virus,HBV)的漏检率 ,探讨荧光定量聚合酶链反应 ( Flurescence quantitative PCR,FQ-PCR)技术用于混合血浆标本病毒核酸检测的可行性。 方法 应用 FQ-PCR技术对常规 ELISA初复检正常献血者 (包括无偿献血者和个体献血者 )的微量血浆汇集池标本 ( 1 0人份× 2 0 μ1 )进行 HBV DNA检测 ,再对阳性汇集池中的标本进行单份检测。用双蒸水和 HBV DNA阴性汇集池中的血浆标本分别对 HBV DNA标准品(浓度为 1 0 3拷贝 /ml)作 1 0～ 5 0倍稀释后行 FQ-PCR测定 ,观察不同的血浆标本混合后是否存在 Taq酶抑制物的叠加作用及对 PCR结果有无影响。对浓度为 1 0拷贝 /ml～ 1 0 4拷贝 /ml的 HBV DNA标准品分别进行 FQ-PCR检测 ,确定试剂盒的敏感度。 结果 1 2 0 0份无偿献血者标本中有 1 1例 HBV DNA阳性 ( 0 .92 % ) ;4 70份个体献血者标本中有 1 0例 HBVDNA阳性 ( 2 .1 3% )。双蒸水与混合血浆稀释的 HBV DNA标准品的 FQ-PCR检测结果 (定性 )完全一致。试剂盒的检出下限为 1 0 2拷贝 /ml。 结论 ELISA法筛查血液存在较高的 HBV漏检率 。

Objective To evaluate the rate of false negative for HBV in blood samples screened by ELISA, and to study the feasibility of FQ PCR in the detection of HBV DNA in pooled samples. Methods Individual donor plasma samples serologically negative for HBsAg, anti HCV, anti HIV detected by ELISA were mixed according to the size of 10×20 μ1. HBV DNA in pooled samples was detected by FQ PCR. Individual donor plasma samples in positive pooled samples were further tested also by FQ PCR. HBV DNA standard controls diluted by dd H 2O and negative pooled samples were detected by FQ PCR so as to understand whether there were inhibitors of Taq DNA polymerase in mixed samples and whether they take effect on the results of FQ PCR. The sensitivity of FQ PCR kit was also tested. Results 11(0.92%) of 1 200 unpaid donators,10(2.13%) of 470 paid donators were positive for HBV DNA. Dd H 2O groups and groups diluted by negative pooled plasma samples showed the same results of FQ PCR. The kit' sensitivity was 10 2 copy/ ml. Conclusion There was higher rate of false negative for HBV in blood samples screened by ELISA. It is feasible to incorporate FQ PCR into ELISA screening blood donors for the nucleic acid of virus.