用PCR扩增及Southern印迹杂交对新疆克拉玛依地区皮肤利什曼病病原体kDNA同源性研究

ANALYSIS OF SEQUENCE HOMOLOGIES IN kDNA OF LEISHMANIA SPECIES CAUSING CUTANEOUS LEISHMANIASIS IN KARAMAY, XINJIANG BY PCR AND SOUTHERN HYBRIDIZATION

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 paper,a 120hp kDNA fragment of Leishamania from cutaneous lesions of 2 parasitologically confirmed patients and 3 Leishmania species (L. tropica, L. infantum, L. gerbdli) were directly amplified with primer 13A and 13B. Southern hybridization was performed with digoxigenin labelled L. tropica, L. infantum, L. gerbilli, L. major kDNA probe separately.Results showed that strong hybridization was found to occur only in CL-PCR-AP with L.tropica kDNA probe, while other Leishmania species kDNA probe showed no detectable signals.Sequence homologies in kDNA of L. infantum and L. donovani Xinjiang isolates with Leishmania species causing cutaneous leishmaniasis have been analysed by using specific L. d primer Ⅰ and Ⅱ. PCR amplified products (297bp) only occurred in L.donovani Sichuan human isolate and L. infantum. 3 samples of CL and L. tropica were negative.Dot hybridization was performed with promastigote of 7 species of Leishmania i.e.L.tropica, L.d Xinjiang 801, L. d Xinjiang 812,4 L. d Xinjiang 901, L. gerbilli, Lizard Leishmania,L.d Gansu isolate with digoxigenin labelled CL-PCR-AP probe. Strong hybridization was found to occur only in L. tropica and positive controls and other Leishmania specises showed absence of any signal. Our results confirmed that homologous sequences exist in kDNA of L.tropica and the species causing CL in Karamay.