PCR检测细粒棘球蚴及DIG标记DNA杂交诊断技术的建立

Detection of Echiococcosis by PCR and the Establishment of Diagnosis Technique by DNA Hybridization Labeled With DIG

目的 :该项研究旨在探讨聚合酶链反应 (PCR)技术在细粒棘球蚴检测和诊断中的应用 ,同时建立Digoxinum(DIG)标记DNA杂交诊断技术。方法 :根据细粒棘球蚴基因片断克隆与序列分析 ,设计了一对特异性引物 ,P15′—GGAATGGAGAGAAGTTAC— 3′,P2 5′—GCAACCTCCGGAACTTGC— 3′。以棘球蚴的囊液、子囊及原头蚴为模板 ,经PCR扩增获得 471bp特异性区带。将扩增产物纯化后 ,用DIG标记DNA ,制备成功特异性核酸探针并用于细粒棘球蚴检测。结果 :经对大肠杆菌、痢疾杆菌、结核分枝杆菌、猪囊尾蚴、健康人白细胞进行PCR扩增和DIG标记DNA探针斑点杂交 ,只有细粒棘球蚴出现单一 471bp特异性区带。PCR的灵敏性为可检出单个原头蚴及 1 0 0— 1 0fg水平的DNA ,而斑点杂交的特异性为 2 5 0 0fg。异源性DNA即使提高点膜量 ,亦不呈现阳性反应。结论 :配以DIG标记的PCR技术在细粒棘球蚴的检测中具有特异、敏感、快速、准确的特点。

Objective:to research the use of PCR in detecting and diagnosing ecninococsus granulosus and conduct a diagnostic technique by DNA hybridization labeled with DIG.Methods:A pairs of primer were designed according to echinococcus granulosus gene fragment clone and sequence analysis special primer as:P1 5'-GGAATGGAGAGAAGTTAC-3',P2 5'-GCAACCTCCGGAACTTGC-3'.The hydatid fluid,secondary hydatid and protoscolex of echinococcus granulosus were used as template,417bp special band was got after being amplified by PCR.The PCR product was purified and labeled with DIG.The special DNA probe was succefully got and can be used to detect the echinococcus granulosus.Result:The DNA from colibacillus;Shigella,tubercle,Cysticercus granulosus and health human leukocyte were extended by PCR and hybrided by DNA probe labeled with DIG,only cystilercus granulosus express 471bp special band.The sensitivity of PCR were that one cystilercus granulosus can be detected or 100-10 fg DNA.while the dot hybridization was 2500fg.Heterologous DNA will not express positive reaction,even increasing the spotting membrane dosages.Conclusions:The sequence of PCR labeled with DIG showed good specificity,high sensitivity,more accuracy and quickness and so it can provide scientific basis to early diagnosis echinococcus granulosus and its epidemiological investigation.