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本研究自新疆克拉玛依地区皮肤利什曼病(CL)患者的皮肤病变组织内抽取微量的利什曼原虫kDNA,采用引物13A、13B进行PCR扩增,获120hP扩增产物(CL-PCR-AP),并转移到尼龙膜上,分别与地高辛(Digoxigenin-11-dUTP)标记的L.tropica、L.infantum、L.gerbilli、L.majorkDNA探针进行Southern印迹杂交,结果可见病变组织内kDNAPCR-AP仅与L.tropicakDNA探针有明显的杂交信号带,提示该地区皮肤利什曼病病原体kDNA与L.tropica有较强的同源性。为进一步分析该病原体与L.infantum和L.donovani新疆各分离株的同源关系,我们采用杜氏利什曼原虫(L.d)种特异引物Ⅰ和Ⅱ对患者病变组织进行PCR扩增,未见扩增产物。用地高辛标记的CL-PCR-AP探针对L.donovani新疆各分离株进行打点杂交,结果显示该地区CL病原体与L.donovani新疆分离株以及其它利什曼原虫呈阴性反应。以上结果证实L.tropicakDNA与克拉玛依地区CL患者的CL-PCR-AP之间的同源关系。
In this study, a small amount of Leishmania kDNA was extracted from skin lesions of skin leishmaniasis (CL) patients in Karamay area of Xinjiang. PCR amplification was carried out using primers 13A and 13B. A 120 hP amplification product (CL-PCR-AP ) And transferred to nylon membrane and labeled with Digoxigenin-11-dUTP. tropica, L. infantum, L. gerbilli, L. Southern blotting was performed using the majork DNA probe. As a result, the kDNA PCR-AP was only found in the diseased tissue. Tropicak DNA probe obvious hybridization signal band, suggesting that the region of skin leishmaniasis pathogen kDNA and L. Tropica has a strong homology. To further analyze the pathogen and L. infantum and L. donovani Xinjiang strains homologous relationship, we use Leishmania donovani (L.d) species-specific primers Ⅰ and Ⅱ lesions of patients with PCR amplification, no amplification products. Ligand-labeled CL-PCR-AP probe pair L. donovani Xinjiang strains were dotted hybridization, the results show that the region CL pathogens and L. donovani Xinjiang isolate and other Leishmania-negative reactions. The above results confirm L. homology between tropicak DNA and CL-PCR-AP in patients with CL in Karamay.