摘要
目的 建立一种检测脑膜炎新型隐球菌聚合酶链反应 (PCR)产物的微孔酶免疫方法 .方法 对 PCR所用 2条引物进行双标记 ,使扩增产物一端标记上生物素 ,另一端标记上地高辛 ,通过包被在微孔板上的亲和素捕获固定 ,然后用碱性磷酸酶标记的地高辛抗体反应后显色测定 .结果 三种 PCR产物检测方法的对照分析表明微孔板酶免疫法敏感性最强 ,加样量 38n L 时即可显色 ,模板 DNA经过 10 1 1倍稀释后仍为阳性结果 ;聚丙烯酰胺凝胶电泳和琼脂糖电泳则分别可检测到 0 .312 5 μL,1.2 5 μL 及 10 7,10 4稀释度 .对临床脑脊液标本的检测结果微孔板酶免疫法和聚丙烯酰胺凝胶电泳的符合率为 10 0 % ,二者敏感性均高于琼脂糖电泳 .结论 建立的方法与常规电泳法相比灵敏准确 ,结果判断客观 ,重复性也较好 ;所测结果可相对反应 PCR产物量的多少 ,此方法可取代电泳法成为常规检测
AIM To establish a micro titer EIA for detecting PCR products of cyrptococcal neoformans. METHODS The primers were modified so that the two terminals of PCR products were labeled with biotin and digoxin respectively.The products were captured by the streptavidin coated in the micro titer wells.Then anti dig antibody AP conjugates were added into the wells. After washing, the NBT BCIP were added and then readings of the micro titers were measured. RESULTS Compared with routine gel electrophoresis, the sensitivity of micro titer EIA was much higher. It could detect precisely even when loaded with 38 nL positive PCR products and after the positive template had been diluted 10 11 times. Polyacrylamide and agarose gel electrophoresis can detect 0.3125 μL,1.25 μL and 10 7,10 4 times respectively. The corresponding rate of EIA and polyacrylamide gel electrophoresis was 100% when 15 clinical samples were detected. CONCLUSION The established PCR EIA was sensitive and accurate.The absorbance of EIA were correlated with the volume of the products.The established method may be used as a routine method for detecting the products of PCR of cyrptococcal neoformans instead of electrophoresis technique.
出处
《第四军医大学学报》
2000年第10期1241-1243,共3页
Journal of the Fourth Military Medical University
基金
陕西省科技发展研究计划资助项目!(1997K12 )
关键词
新型隐球菌
聚合酶链反应
微孔酶免疫法
cyrptococcal neoformans
polymerase chain reaction
enzyme immunoassay
