Lawrie J M, Jackson P R, Stiteler J M, Hockmeyer W T
Am J Trop Med Hyg. 1985 Mar;34(2):257-65. doi: 10.4269/ajtmh.1985.34.257.
We report the characterization of Leishmania (L. infantum, L. donovani, and L. major) kinetoplast DNA (kDNA) by the use of restriction endonuclease digestion patterns and Southern hybridizations. Overall, the sizes and fragment patterns of MspI restriction endonuclease-produced DNA fragments vary from species to species. However, kDNA isolates from different species and strains cross-reacted to a great extent in Southern hybridization experiments. Only kDNA isolated from L. infantum and L. major had little homology during hybridization reactions. To prepare DNA probes that would differentiate between species of Leishmania, minicircle kDNA was digested with restriction enzymes and ligated to an E. coli plasmid. Several plasmids were isolated that specifically detect in hybridization experiments as few as 5 X 10(3) L. donovani or L. infantum promastigotes lysed on nitrocellulose filters.
我们报告了利用限制性内切酶消化模式和Southern杂交对利什曼原虫（婴儿利什曼原虫、杜氏利什曼原虫和硕大利什曼原虫）动质体DNA（kDNA）进行的特征分析。总体而言，MspI限制性内切酶产生的DNA片段的大小和片段模式因物种而异。然而，来自不同物种和菌株的kDNA分离物在Southern杂交实验中发生了很大程度的交叉反应。在杂交反应中，只有从婴儿利什曼原虫和硕大利什曼原虫分离的kDNA具有很少的同源性。为了制备能够区分利什曼原虫物种的DNA探针，将微小环kDNA用限制性酶消化并连接到大肠杆菌质粒上。分离出了几种质粒，它们在杂交实验中能够特异性地检测到在硝酸纤维素滤膜上裂解的低至5×10³个杜氏利什曼原虫或婴儿利什曼原虫前鞭毛体。
