杜氏利什曼原虫特异性kDNA片段扩增及用于内脏利什曼病诊断的研究  

AMPLIFICATION OF A kDNA FRAGMENT WITH L.DONOVANI SPECIES-SPECIFICITY BY PCR AND APPLICATION IN VISCERAL LEISHMANIASIS DIAGNOSIS

作　　者：杨文天[1] 胡孝素[1] 

机构地区：[1]华西医科大学寄生虫学研究室,成都610041

出　　处：《实用寄生虫病杂志》1993年第3期1-4,共4页Journal of Practical Parasitic Diseases

基　　金：纽约中华医学基金会;国家教委博士点专项基金

摘　　要：本文报道了用杜氏利什曼原虫特异的一对寡核苷酸引物Ⅰ和Ⅱ进行PCR,扩增微环kDNA分子上一种特异性kDNA片段,进行杜氏利什曼原虫虫种鉴定和病原体检测。敏感性分析表明用此法能够检测到的最低模板DNA需要量为1fg,检测健康犬全血稀释的利什曼原虫前鞭毛体,最低可达2个/ml。扩增六种不同的利什曼原虫kDNA样品,在L.donovani四川人株、四川犬株和L.infantum出现阳性产物,其长度和设计扩增长度一致,扩增产物和地高辛标记的重组质粒探针pLK2斑点杂交结果表明为利什曼原虫kDNA序列。应用此对引物,检测8份内脏利什曼病患者骨髓样品和4份血清样品,经琼脂糖凝胶电泳,Southern杂交证实,分别有7例和2例阳性,显示出可喜的应用前景。Using a set of oligonucleotide primers I and II, PCR was conducted to amplify a minicircle kDNA fragment(297 bp) for identification and detection of Leishmania pathogens. Sensitivity analysis showed that the minimal template kDNA detected is as low as 1 fg. When L. dono- vani (MHOM/CN/86/SC6) promastigotes were serially diluted with normal canine blood, about 2 promastigotes/ml can be detected after 25 to 30 cycle PCR amplification. Amplifying the kDNAs from L. donovani Sichuan human isolate, Sichuan canine isolate, L. infantum, L. mexicana, L. briziliensis, L. major, lizard Leishmania, positive products can be visulized only in L. donovani Sichuan human isolate, Sichuan canine isolate and L. infantun, after elec- trophoresis on agarose gel and staining with E.B. Dot hybridization of the amplified products with the recombinant plasmid pLK2 labeled with digoxigenin-11-dUTP confirmed that they are Leishmania sequences. Based on this set of primers, 8 bone marrow samples and 4 serum samples from the confirmed visceral leishmaniasis patients were examined, and 7 and 2 were positive respectively. This result was also approved by Southern hybridization with the same probe above. The result of this study shows that diagnosis of visceral leishmaniasis based on detecting kDNA in peripheral blood by PCR amplification is full of promising.

关 键 词：内脏利什曼病 聚合酶链反应 kDNA 

分 类 号：R531.6[医药卫生—内科学]