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作者从已建立的问号钩体基因文库中筛选合成一对引物G_1G_2,中国四川14份早期钩体病患者血清中的DNA经硫酸氰胍、二氧化硅和蛋白酶K纯化后,在G_1G_2引导下行聚合酶链反应(PCB),并以地高辛标记的同源探针行DNA印迹杂交,全部血清样品均获得确切地阳性结果。用地高辛—硷性磷酸酶发光体标记PCR扩增的同源DNA探针,与16株Yasuda基因组钩体作DNA印迹分析,其中8株存在同源单拷贝序列,它们皆为我国常见和流行的优势致病菌株,本研究结果表明:引物G_1G_2可用于四川钩体PCR检测和分析,PCR常规结合同源DNA探针杂交识别,能提高其敏感性和可靠性;地高辛—硷性磷酸酶发光体配合PCR等方法,可使微量DNA的检测和分析更趋完善。同时,本研究还提供了有价值的钩体DNA同源序列资料。
The authors screened and synthesized a pair of primers G_1G_2 from the established library of Leptospira interrogans (Leptospira interrogans) genes. The DNA of sera from 14 patients with early leptospirosis in Sichuan, China was purified by GAG, silica and proteinase K and then polymerized under G_1G_2 Enzyme-linked immunosorbent assay (PCB) and hybridization with digoxigenin-labeled homologous probes by Southern blotting, all serum samples obtained positive results. The homologous DNA probes amplified by digoxigenin-alkaline phosphatase were labeled with 16 Yasuda genomes for Southern blot analysis. Among them, 8 were homologous single-copy sequences, all of which were common and epidemic in our country The results showed that the primer G_1G_2 could be used for PCR detection and analysis of Leptospira interrogans in Sichuan Province. PCR was used to detect the homology DNA probe hybridization, which could increase the sensitivity and reliability of digoxin. Digoxigenin-alkaline phosphatase Enzyme luminescence with PCR and other methods, the detection and analysis of trace DNA can be more perfect. At the same time, this study also provided valuable information of homologous sequences of leptospira DNA.