Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested the existence of two clades of amphibian Perkinsea: a putatively non-pathogenic clade linked to asymptomatic infections of tadpoles in Africa, Europe and South America, and a putatively pathogenic clade linked to disease and mass mortality events of tadpoles in North America. Here, we describe the development of a duplex TaqMan qPCR assay to detect and discriminate between rDNA sequences from both clades of Perkinsea in amphibian tissues. The assay uses a single primer pair to target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared between the two clades, and two dual-labelled probes to target a region within this fragment that is diagnostic for each clade. This assay enables rapid screening for each of the two Perkinsea groups, allowing for detection, primarily of the phylogenetic group associated with disease outbreaks, and secondarily for the phylogenetic group with no current disease relationship identified. Incorporation of our novel qPCR assay into the routine surveillance of amphibian populations will allow for the assessment of the incidence of each protist clade, thereby providing an improved understanding of Perkinsea infection pervasiveness and a method to underpin future conservation planning.

已发现珀金西门中的空泡质子传播者在广泛的分类学和地理范围内感染两栖动物。系统发育分析表明存在两栖两栖类珀金西氏进化枝：一个与非洲，欧洲和南美的t无症状感染有关的非病原性进化枝，以及与北美disease的疾病和大规模死亡事件有关的假定病原性进化枝。 。在这里，我们描述了一种双链TaqMan qPCR检测方法的开发，以检测和区分两栖类组织中来自珀金西两个进化枝的rDNA序列。该测定法使用单个引物对靶向两个进化枝之间共享的18S小亚基（SSU）核糖体RNA（rRNA）基因区域，并使用两个双标记探针靶向此片段内可诊断每个进化枝的区域。该测定法能够快速筛查两个Perkinsea组中的每个组，从而主要检测与疾病暴发相关的系统发育组，其次检测未发现当前疾病关系的系统发育组。将我们新颖的qPCR检测方法纳入两栖动物种群的常规监测中，可以评估每个原生生物进化枝的发生率，从而更好地了解Perkinsea感染的普遍性，并为今后的保护规划提供基础。