【摘要】 滚环复制是噬菌体繁殖所采取的一种基因复制方式,这种方式可使单链的环形分子在聚合酶和引物的作用下进行体外自我扩增。本文中用可特异性连接环化的寡核苷酸链作为探针,分别进行了1份细胞培养的禽流感病毒H5N1亚型样品、1份细胞培养的SARS病毒样品和4份丙型肝炎病毒阳性血清样品的检测。检测原理是探针与靶序列杂交后便可在T4DNA连接酶的作用下形成滚环复制中的环化单链分子,该分子在同温下可被特异性引物滚动复制和支链扩增。本文还利用按禽流感病毒NA1基因区序列合成的模拟DNA分子对该检测方法的灵敏度进行了测试。结果显示:利用固相RCA技术成功检测到三种RNA病毒的基因,该方法的灵敏度可达到能检测103拷贝模式DNA分子的水平。与传统的PCR方法敏感性的比较尚待进一步研究。更多还原

【Abstract】 Rolling circle amplification is a replicative style of bacteriophage. The ssDNA circle can isothermally self-replicate by this mechanism. In this study, HCV positive serum samples, cell-cultured avian influenza virus subtype H5N1 and SARS virus samples were used to evaluate the detection ability of rolling circle amplification with circle probes, which can hybridize with target molecules and be circularized by T₄ DNA ligase. Then, the circularized probe can be amplified by an isothermal ramification amplification strategy with primers and exo-Bst DNA polymerase. We have also tested sensitivity of this method by serial 10-fold dilutions of synthetical DNA template molecules （10⁷to10²molecules）.The results showed that the method can detect successfully three different RNA viruses including avian influenza virus subtype H5N1,HCV and SARS viruses. Additionally, the sensitivity test demonstrated that rolling circle amplification method achieves a sensitivity of 10³ template DNA molecules.更多还原