PCR与DNA杂交技术在细粒棘球蚴诊断方面的实验研究  

EXPERIMENTAL RESEARCH WITH PCR AND DNA HYBRIDIZATION TECHNIQUE IN DETECTION AND DIAGNOSIS OF ECHINOCOCCUS GRANULOSUS

作　　者：冯笑梅[1] 冀虎岗[1] 曹银芳[1] 王文灏[1] 

机构地区：[1]内蒙古自治区医院,呼和浩特010017

出　　处：《中国血吸虫病防治杂志》2004年第3期206-209,共4页Chinese Journal of Schistosomiasis Control

基　　金：内蒙古自治区科学技术委员会资助课题 ( No.960 12 4)

摘　　要：目的　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 ,建立 DIG标记DNA杂交诊断技术。方法　根据细粒棘球蚴基因片段克隆与序列分析 ,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 4 71bp特异性区带。将扩增产物纯化后 ,用 DIG标记 DNA ,将制备成的特异性核酸探针用于细粒棘球蚴检测。结果　经对大肠杆菌、粪肠杆菌、结核分支杆菌、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 4 71bp特异性区带。 PCR的灵敏性为检出单个原头蚴及 10 0～ 10 fg水平的 DNA,而斑点杂交的敏感性为 2 5 0 0 fg。异源性 DNA即使提高点膜量 ,亦不出现阳性反应。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 explore the application of PCR in detection and diagnosis of Echinococcus granulosus and develop a diagnostic technique by DNA hybridization labeled with DIG.Methods A pair of primers were designed according to Echinococcus granulosus gene fragment sequence, and the special primers were as: P1 5′-GGAATGGAGAGAAGTTAC-3′ P2 5′-GCAACCTCCGGAACTTGC-3′. The hydatid fluid, secondary hydatid and protoscolex of Echinococcus granulosus were used as template, 417 bp special band was got after being amplified by PCR. The PCR product was purified and labeled with DIG. The special DNA probe was successfully got and was used to detect the Echinococcus granulosus. Results The DNA from Escherichia coli, Shigella, Tubercle, Cysticercus granulosus and healthy human leukocyte were extended by PCR and hybrided by DNA probe labeled with DIG, and only Cysticercus granulosus expressed 471 bp special band. The sensitivity of PCR was that one Cysticercus granulosus can be detected or 100-10 fg DNA, while the dot hybridization was 2500 fg. Heterologous DNA did not be positive reaction, even increasing the spotting membrane dosages. Conclusion The technique of PCR labeled with DIG showed good specificity, high sensitivity, more accuracy and quickness and so it can provide scientific basis for early diagnosis and epidemiological investigation of Echinococcus granulosusEXPERIMENTAL RESEARCH WITH PCR AND DNA HYBRIDIZATION TECHNIQUE IN DETECTION AND DIAGNOSIS OF ECHINOCOCCUS GRANULOSUS$$$$ Feng Xiaomei, Ji Hugang, Cao Yinfang, Wang Wenhao (Department of Clinical Laboratory, Inner Mongolia Autonomous Region Hospital, Huhhot 010017, China) Abstract Objective To explore the application of PCR in detection and diagnosis of Echinococcus granulosus and develop a diagnostic technique by DNA hybridization labeled with DIG.Methods A pair of primers were designed according to Echinococcus granulosus gene fragment sequence, and the special primers were as: P1 5′-GGAATGGAGAGAAGTTAC-3′ P2 5′-GCAACCTCCGGAACTTGC-3

关 键 词：聚合酶链反应 细粒棘球蚴 核酸杂交探针 

分 类 号：R532.32[医药卫生—内科学]