Uliana S R, Nelson K, Beverley S M, Camargo E P, Floeter-Winter L M
Departamento de Parasitologia ICB USP, São Paulo, Brazil.
J Eukaryot Microbiol. 1994 Jul-Aug;41(4):324-30. doi: 10.1111/j.1550-7408.1994.tb06085.x.
A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5' and 3' termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (> 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non-radioactive labeling showed the same specificity and sensitivity as radioactive probes.
本文描述了一种基于小亚基核糖体DNA序列差异鉴别利什曼原虫的方法。该方法是使用针对小亚基核糖体RNA 5'和3'末端的保守引物，通过聚合酶链反应扩增整个2.2 kb的小亚基rDNA，然后将产物点样到尼龙膜上，与标记的寡核苷酸杂交。杂交探针的设计基于亚马逊利什曼原虫、硕大利什曼原虫和圭亚那利什曼原虫的完整小亚基rDNA序列以及墨西哥利什曼原虫、巴西利什曼原虫、热带利什曼原虫和恰加斯利什曼原虫的部分序列。发现不同物种之间存在高度的序列相似性（>99%）。然而，也出现了足够的序列差异，从而能够设计出针对物种复合体的内部寡核苷酸探针。该方法成功地区分了多种利什曼原虫分离株。该方法能检测到低至10个培养的生物体，并能在实验感染动物的组织样本中检测到寄生虫。非放射性标记显示出与放射性探针相同的特异性和灵敏度。
