Universidade Federal do Pampa - UNIPAMPA, Uruguaiana, RS, Brasil.
Universidade Federal de Ciências da Saúde de Porto Alegre - UFCSPA, Porto Alegre, RS, Brasil.
Rev Bras Parasitol Vet. 2022 Jul 18;31(3):e005222. doi: 10.1590/S1984-29612022035. eCollection 2022.
The aim of this study was to characterize Leishmania spp. from canine and feline samples using Polymerase Chain Reaction (PCR)- Restriction Fragment Length Polymorphism (RFLP). It was conducted in the southern region of Brazil, located at border crossings to Argentina and Uruguay. Samples were collected from 116 dogs (Canis lupus familiaris) and 89 cats (Felis catus). The PCR was performed to screen for an LT1 fragment from kinetoplast DNA (kDNA) target gene, and positive samples were subjected to a second PCR for an internal transcribed spacers (ITS1) region from ribosomal DNA (rDNA) target. RFLP was performed using the Haemophilus aegyptius (HAE III) restriction endonuclease (Fermentas ®). Positive samples by PCR ITS1 were sequenced and deposited in NCBI GenBank, and a phylogenetic analysis was developed. We found that 12.9% (15/116) of the samples from dogs were positive. All the 89 cat samples were negative. Positive samples were tested against Leishmania reference strains presenting different patterns in PCR-RFLP, and these samples showed bands denoting similarity to the standard species of Leishmania infantum, proven through sequencing and phylogenetic analysis. The RFLP technique, alone, was shown to be feasible for practical application and confirmation of the involved Leishmania spp.
本研究旨在使用聚合酶链反应（PCR）-限制性片段长度多态性（RFLP）对来自犬和猫样本的利什曼原虫进行特征分析。该研究在巴西南部进行，位于与阿根廷和乌拉圭接壤的边境过境点。采集了 116 只犬（Canis lupus familiaris）和 89 只猫（Felis catus）的样本。进行 PCR 以筛选动质体 DNA（kDNA）靶基因的 LT1 片段，阳性样本进行第二轮 PCR 以筛选核糖体 DNA（rDNA）靶基因的内部转录间隔区（ITS1）区域。使用埃及嗜血杆菌（HAE III）内切酶（Fermentas ®）进行 RFLP。对 PCR ITS1 阳性样本进行测序并在 NCBI GenBank 中进行了保存，同时还进行了系统发育分析。结果发现，12.9%（15/116）的犬样本为阳性。89 只猫样本均为阴性。对阳性样本进行了针对不同 PCR-RFLP 模式的利什曼原虫参考株的检测，这些样本通过测序和系统发育分析显示与利什曼原虫婴儿种标准种具有相似性。RFLP 技术本身可用于实际应用和确认涉及的利什曼原虫。
