Department of Biomedical Sciences and Veterinary Public Health, Section for Parasitology, Swedish University of Agricultural Sciences, P.O. Box 7036, Uppsala, Sweden.
Parasitol Res. 2019 Oct;118(10):2901-2907. doi: 10.1007/s00436-019-06414-7. Epub 2019 Aug 6.
Cooperia sp. and Ostertagia sp. are two cosmopolitan parasitic nematodes often found in mixed gastrointestinal infections in cattle across temperate regions. In light of the recent increase in the emergence of anthelmintic resistance in these and other nematodes derived from cattle around the globe, and their negative impact on animal health and productivity, novel molecular assays need to be put forth in order to facilitate the monitoring of parasite burden in infected herds, using pasture and/or fecal samples. Here, we describe a novel droplet digital PCR platform-based concept for precise identification and quantification of the two most abundant and important parasite genera in grazing western European cattle. By exploiting a single nucleotide difference in the two parasites' ITS2 sequence regions, we have developed two specific hydrolysis probes labeled with FAM™ or HEX™ fluorophores, which can not only distinguish between the DNA sequences of the two, but also quantify them in mixed DNA samples. A third, newly developed universal probe was also tested along the genus-specific probes to provide a robust and accurate reference. It was evident that the universal probe displayed congruent results to those obtained by the genus-specific probes when used with DNA from both parasites in a single sample. All in all, the results of our assay suggest that this novel protocol could be used to distinguish and quantify cattle parasites belonging to the two most important genera (i.e., Cooperia and Ostertagia) in a single mixed DNA sample.
无尾感器纲线虫（Cooperia sp.）和奥斯特利线虫（Ostertagia sp.）是两种世界性寄生线虫，通常在温带地区牛的混合胃肠道感染中发现。鉴于这些线虫以及来自全球牛的其他线虫的驱虫剂耐药性最近有所增加，并且对动物健康和生产力产生负面影响，需要提出新的分子检测方法，以便利用牧场和/或粪便样本来监测受感染牛群中的寄生虫负担。在这里，我们描述了一种基于液滴数字 PCR 平台的新型概念，用于精确鉴定和定量放牧的西欧牛中两种最丰富和最重要的寄生虫属。通过利用两种寄生虫 ITS2 序列区域中的单个核苷酸差异，我们开发了两种带有 FAM™或 HEX™荧光染料标记的特异性水解探针，不仅可以区分两种寄生虫的 DNA 序列，而且可以在混合 DNA 样本中对其进行定量。还测试了第三种新开发的通用探针与属特异性探针一起使用，以提供稳健而准确的参考。显然，当将通用探针与单个样本中的两种寄生虫的 DNA 一起使用时，它与属特异性探针的结果一致。总而言之，该检测方法的结果表明，该新方案可用于在单个混合 DNA 样本中区分和定量属于两个最重要属（即 Coopria 和 Ostertagia）的牛寄生虫。
