Fürnkranz Ursula, Walochnik Julia, Grimm Felix, Deplazes Peter, Aspöck Horst
Abteilung für Medizinische Parasitologie, Klinisches Institut für Hygiene und Medizinische Mikrobiologie der Medizinischen Universität Wien, Wien, Osterreich.
Wien Klin Wochenschr. 2004;116 Suppl 4:30-4.
Leishmania spp. are the causative agents of visceral, cutaneous and mucocutaneous leishmaniosis with several taxa ("species") of the genus Leishmania being involved in human disease. As diagnostics based on microscopical detection of the parasites or on serological tests are often unsatisfactory, also molecular biological methods, particularly the polymerase chain reaction (PCR), have been employed for the detection of Leishmania spp. in the past years. The aim of the present study was to compare different PCR-protocols and optimise them for our needs, placing emphasis on the improvement of DNA isolation. PCR was performed with whole cell DNA isolated from cultures, as well as from simulated blood samples and clinical samples. Three different methods for the isolation of DNA from blood samples and two different PCR-protocols, one amplifying a fragment of the 18S rDNA and one for the amplification of the whole kDNA-circle, were applied and compared. No significant difference in sensitivity was detected between the different PCR-protocols, however, it was shown that the highest yield of DNA was achieved with a DNA isolation protocol based on urea.
利什曼原虫属是内脏利什曼病、皮肤利什曼病和黏膜皮肤利什曼病的病原体，该属的几个分类单元（“种”）会引发人类疾病。由于基于显微镜检测寄生虫或血清学检测的诊断方法常常不尽人意，因此在过去几年中，分子生物学方法，尤其是聚合酶链反应（PCR），也被用于检测利什曼原虫属。本研究的目的是比较不同的PCR方案，并根据我们的需求对其进行优化，重点是改进DNA提取方法。PCR使用从培养物、模拟血样和临床样本中分离的全细胞DNA进行。应用并比较了三种从血样中分离DNA的不同方法和两种不同的PCR方案，一种扩增18S rDNA片段，另一种扩增整个kDNA环。不同的PCR方案之间未检测到敏感性的显著差异，然而，结果表明基于尿素的DNA提取方案可获得最高的DNA产量。
