Eisenberger C L, Jaffe C L
Department of Parasitology, The Kuvin Centre for the Study of Infectious and Tropical Diseases, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Exp Parasitol. 1999 Jan;91(1):70-7. doi: 10.1006/expr.1999.4355.
We have developed a permissively primed intergenic polymorphic-polymerase chain reaction (PPIP-PCR) which distinguishes between the Old World Leishmania complexes L. major, L. tropica, L. donovani, and L. aethiopica. This technique pairs one parasite-specific and one nonspecific oligonucleotide primer for the PCR. The specific primer was chosen from a unique leishmanial DNA sequence, clone pDOG 2, isolated from a L. donovani chagasi genomic DNA expression library. This sequence has a high DNA homology to the intergenic region of the L. major B/C genes which belong to the polymorphic LmcDNA16 gene family. The specific intergenic primer contains a high GC content, a stem-loop, and a 3'-CG residue. The nonspecific primer was selected from within the pBluescript (SK) plasmid. Using PPIP-PCR, parasites belonging to the L. major, L. tropica, L. donovani, and L. aethiopica complexes could be easily identified directly following agarose gel electrophoresis by the simple profiles of their PCR products. In addition, it was possible to discriminate between strains of L. major or L. donovani from distant geographical regions. Amplification of genomic DNA isolated from several nonleishmanial kinetoplastids yielded either no PCR products or unique bands which were distinct from the leishmanial profiles. Genomic DNA from nonkinetoplastid parasites, plants, or mammals was not amplified by PPIP-PCR. This technique is a rapid and reproducible method for the characterization of Old World Leishmania.
我们开发了一种宽松引物基因间多态性聚合酶链反应（PPIP-PCR），可区分旧大陆利什曼原虫复合体中的硕大利什曼原虫、热带利什曼原虫、杜氏利什曼原虫和埃塞俄比亚利什曼原虫。该技术在PCR中使用一对寄生虫特异性和非特异性寡核苷酸引物。特异性引物选自从杜氏利什曼原虫恰加斯基因组DNA表达文库中分离出的独特利什曼原虫DNA序列克隆pDOG 2。该序列与属于多态性LmcDNA16基因家族的硕大利什曼原虫B/C基因的基因间区域具有高度DNA同源性。特异性基因间引物含有高GC含量、一个茎环和一个3'-CG残基。非特异性引物选自pBluescript（SK）质粒内部。使用PPIP-PCR，通过琼脂糖凝胶电泳后其PCR产物的简单图谱，可轻松直接鉴定属于硕大利什曼原虫、热带利什曼原虫、杜氏利什曼原虫和埃塞俄比亚利什曼原虫复合体的寄生虫。此外，还能够区分来自遥远地理区域的硕大利什曼原虫或杜氏利什曼原虫菌株。从几种非利什曼动基体分离的基因组DNA扩增，要么没有PCR产物，要么产生与利什曼原虫图谱不同的独特条带。PPIP-PCR未扩增非动基体寄生虫、植物或哺乳动物的基因组DNA。该技术是一种快速且可重复的旧大陆利什曼原虫鉴定方法。
