一种用于定量利什曼病小鼠模型中寄生虫负荷的实时聚合酶链反应检测法。
A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis.
作者信息
Antonia Alejandro L, Wang Liuyang, Ko Dennis C
机构信息
Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA.
Division of Infectious Diseases, Department of Medicine, Duke University, Durham, NC, USA.
出版信息
PeerJ. 2018 Nov 9;6:e5905. doi: 10.7717/peerj.5905. eCollection 2018.
Eukaryotic parasites in the genus place approximately 350 million people per year at risk of disease. In addition to their global health significance, spp. have served as an important model for delineating basic concepts in immunology such as T-helper cell polarization. There have been many qPCR-based assays reported for measuring parasite burden in humans and animals. However, these are largely optimized for use in clinical diagnosis and not specifically for animal models. This has led several of these assays to have suboptimal characteristics for use in animal models. For example, multi-copy number genes have been frequently used to increase sensitivity but are subject to greater plasticity within the genome and thus may confound effects of experimental manipulations in animal models. In this study, we developed a sybr-green based quantitative touchdown PCR assay for a highly conserved and single-copy putative RNA-binding protein, DRBD3. With primers that share greater than 90% sequence identity across all sequenced spp., we demonstrate that this assay has a lower limit of detection of 100 fg of parasite DNA for , , , and . Using C57BL6/J mice, we used this assay to monitor parasite burden over 1 month of infection with two strains of (Seidman and Friedlin), and These characteristics rival the sensitivity of previously reported qPCR based methods of parasite quantitation while amplifying a stable, single copy gene. Use of this protocol in the future will lead to improved accuracy in animal based models and help to tease apart differences in biology of host-parasite interactions.
该属的真核寄生虫每年使约3.5亿人面临患病风险。除了具有全球健康意义外,该属物种还作为一个重要模型,用于阐述免疫学中的基本概念,如T辅助细胞极化。已经有许多基于qPCR的检测方法被报道用于测量人和动物体内的寄生虫负担。然而,这些方法大多是为临床诊断而优化的,并非专门针对动物模型。这导致其中一些检测方法在动物模型中的应用具有次优特性。例如,多拷贝数基因经常被用于提高灵敏度,但在基因组内具有更大的可塑性,因此可能会混淆动物模型中实验操作的效果。在本研究中,我们针对一种高度保守的单拷贝假定RNA结合蛋白DRBD3,开发了一种基于SYBR Green的定量降落PCR检测方法。使用在所有已测序的该属物种中序列同一性大于90%的引物,我们证明该检测方法对该属的几种物种(未提及具体物种名称)的寄生虫DNA检测下限为100 fg。使用C57BL6/J小鼠,我们使用该检测方法监测了感染两种该属菌株(Seidman和Friedlin,未提及具体感染的寄生虫物种名称)1个月期间的寄生虫负担。这些特性可与先前报道的基于qPCR的寄生虫定量方法的灵敏度相媲美,同时扩增的是一个稳定的单拷贝基因。未来使用该方案将提高基于动物的模型的准确性,并有助于区分宿主 - 寄生虫相互作用生物学中的差异。