聚合酶链反应技术检测弓形虫核酸的研究

STUDIES ON THE DETECTION OF DNA OF TOXOPLASMAGONDII BY POLYMERASE CHAIN REACTION

本文通过克隆的ＲＨ株弓形虫核酸ＴＧＲ４片段，设计并合成了一对长度为２０ｂｐ的寡核苷酸引物，扩增靶序列长度为７７８ｂｐ，建立了聚合酶链反应技术检测弓形虫核酸的特异敏感方法。检测７株弓形虫株及临床疑似弓形虫病患者样本，均出现特异扩增带，而其它８种病原体以及正常人及动物的ＤＮＡ均未见特异扩增带。扩增产物用地高辛素标记作探针，与以上出现特异扩增带的ＤＮＡ模板能斑点杂交，而与无扩增带的其余模板ＤＮＡ无斑点杂交。结果显示，该引物具有高度的保守性及特异性。在含１０５个人白细胞中，可检测到２个弓形虫ＤＮＡ或１ｐｇＤＮＡ。并通过人工感染弓形虫鼠血样品的检测表明，所建立的聚合酶链反应体系具有高度的敏感性，能早期检出弓形虫感染样品，在弓形虫病的临床诊断中具有重要的应用价值。

A technique using polymerase chain reaction to amplify specific fragment of Toxoplasma gondii DNA had been established. According to TGR4 fragment of RH strain DNA of Toxoplasma gondii, two primers which are 20 base pairs in length were designed respectively. The results demonstrated that the primers are conserved and presented in all strains of Toxoplasma gondii tested to date. DNAs of a variety of organisms, for instance, Plasmodium berghei, were not amplified in the reactive systems. In the presence of 105 human leukocytes, as few as two parasites could be detected after 3 5 cycles'amplifieation. The resultant fragments (778bp) were labeled with digoxigenin as a probe and Dot-blot hybridizated with DNAs of seven Toxoplasma gondii strains tested which were found positive, while that of some other organisms tested were negative.