PCR-微流芯片检测HBV DNA在血液筛查中的初步应用

HBV DNA assay using PCR-microfluidic chips for blood screening

目的 探讨在血液筛查中检测HBVDNA的必要性及应用PCR 微流芯片检测献血者HBVDNA可运 行模式。方法用PCR 微流芯片检测已经过常规ELISA初、复检全项测定合格献血者的微量血清汇集池(5人 份×50μl)HBVDNA,再对阳性血清汇集池中的标本进行单份检测。用HBVDNA(-)的献血者血清系列稀释 HBVDNA标准质控血清,分别测定其含量,以确定该方法的灵敏度;分别检测37例HBeAg(+)、16例HCV RNA(+)、3例抗 HAVIgM(+)患者标本的HBVDNA,观察其特异性;再重复检测不同稀释度的HBVDNA标 准质控血清,以了解其批内、批间变异。结果 545份(分109个血清汇集池)无偿献血标本中有7份HBVDNA阳 性(1.28%)。PCR 微流芯片法检测HBVDNA敏感度为4.81×102拷贝/ml;HBeAg(+)患者的HBVDNA阳性 检出率为100%(37/37),而HCVRNA(+)、抗 HAVIgM(+)患者的HBVDNA全为阴性;批间、批内变异范围 分别为15.6%～40.2%、11.9%～30.6%。结论无偿献血者开展HBVDNA检测是必要的。PCR 微流芯片法 具有操作简便、快速、敏感度高、特异性强、重复性好等特点,把它应用于血液筛查中并利用混合标本检测HBV DNA是可行的,这将有助于进一步提高输血的安全性。

Objective To test HBV DNA by using PCR-microfluidic chip assay. Methods Pooled sera ( 5×50ul ) negative for ELISA serological tests were tested for HBV DNA using PCR-microfluidic chips assay. Individual donor samples were tested if the pooled sera were positive. The sensitivity of PCR-microfluidic chips assay was determined by serial dilutions of the standard control serum. The specificity of PCR-microfludic chips assay was also determined by testing 56 various serum samples. Serial dilutions of the standard control sera were tested repeatedly for understanding the inter- and intra-assay variation of this method. Results Seven of 545 nonrenumerated donors (1.28%) were found positive for HBV DNA. The sensitivity of PCR-microfluidic chips assay was 4.81×102copies/ml. The HBV DNA was positive for all 37 samples from HBeAg positive patients. The HBV DNA tests of samples from HCV RNA positive patients, anti-HAV IgM positive patients were all negative. The inter- and intra assay CV ranges were 15.6%~40.2% and 11.9%~30.6% respectively. Conclusion It is necessary to test HBV DNA for improving blood safety and it is feasible to test pooled serum samples for HBV DNA by PCR-microfluidic chips assay, because it is convenient, time-saving, sensitive, specific and the results are reproducible.