Bifeld Eugenia
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
altona Diagnostics GmbH, Hamburg, Germany.
Methods Mol Biol. 2019;1971:249-263. doi: 10.1007/978-1-4939-9210-2_13.
While infecting humans and other mammals, Leishmania spp. are obligate intracellular parasites. Therefore, for the purpose of therapeutic intervention and the study of infectivity, the relevant form of Leishmania spp. is the intracellular amastigote. Therefore, monitoring intracellular parasite load is an essential requirement in many fields of Leishmania research. Real-time quantitative PCR is a highly accurate technique for the detection and quantification of parasite burden in in vitro or in vivo infection experiments. The quantification of DNA for standard curves shows linearity over a 5 to 6-log concentration range indicating the high sensitivity of the method. Moreover, qPCR allows for the simultaneous quantification of host and parasite DNA in the same reaction, thereby allowing for an assessment of relative parasite load for basic research, but also for low- to medium-throughput compound screening. The method also allows to analyze late stages of in vitro infections where host cells and parasites have detached from surfaces and escape microscopy-based assays.
利什曼原虫属感染人类和其他哺乳动物时，是专性细胞内寄生虫。因此，出于治疗干预和感染性研究的目的，利什曼原虫属的相关形态是细胞内无鞭毛体。所以，监测细胞内寄生虫载量是利什曼原虫研究许多领域的一项基本要求。实时定量PCR是用于检测和定量体外或体内感染实验中寄生虫负荷的一种高度准确的技术。用于标准曲线的DNA定量在5至6个对数浓度范围内呈线性，表明该方法具有高灵敏度。此外，qPCR允许在同一反应中同时定量宿主和寄生虫DNA，从而不仅可用于基础研究中相对寄生虫载量的评估，还可用于低至中等通量的化合物筛选。该方法还能够分析体外感染的后期阶段，此时宿主细胞和寄生虫已从表面脱离，无法通过基于显微镜的检测方法进行检测。
