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Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani

机译：液滴数字PCR技术检测微量巴氏杆菌和邓巴氏杆菌的开发

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摘要

Babesia spp. are obligate protozoan parasites of red blood cells. Transmission to humans occurs through bites from infected ticks or blood transfusion. Infections with B. microti account for the majority of the reported cases of human babesiosis in the USA. A lower incidence is caused by the more recently described species B. duncani. The current gold standard for detection of Babesia is microscopic examination of blood smears. Recent PCR-based assays, including real-time PCR, have been developed for B. microti. On the other hand, molecular assays that detect and distinguish between B. microti and B. duncani infections are lacking. Closely related species of Babesia can be differentiated due to sequence variation within the internal transcribed spacer (ITS) regions of nuclear ribosomal RNAs. In the present study, we targeted the ITS regions of B. microti and B. duncani to develop sensitive and species-specific droplet digital PCR (ddPCR) assays. The assays were shown to discriminate B. microti from B. duncani and resulted in limits of detection of ~10 gene copies. Moreover, ddPCR for these species were useful in DNA extracted from blood of experimentally infected hamsters, detecting infections of low parasitemia that were negative by microscopic examination. In summary, we have developed sensitive and specific quantitative ddPCR assays for the detection of B. microti and B. duncani in blood. Our methods could be used as sensitive approaches to monitor the progression of parasitemia in rodent models of infection as well as serve as suitable molecular tests in blood screening.
机译：巴贝虫属是红细胞的专性原生动物寄生虫。通过感染的s虫叮咬或输血发生向人类的传播。在美国，报告的大多数人类杆状杆菌病病例都感染了小肠杆菌。发生率较低的原因是最近描述的物种敦煌白僵菌。当前检测巴贝虫病的金标准是血液涂片的显微镜检查。已经针对微量芽孢杆菌开发了基于PCR的最新测定法，包括实时PCR。另一方面，缺乏检测和区分小肠芽孢杆菌和敦煌芽孢杆菌感染的分子测定法。由于核糖体RNA内部转录间隔区（ITS）区域内的序列变异，可以区分密切相关的巴贝斯虫。在当前的研究中，我们针对B.microti和B.duncani的ITS区域开发了敏感的和物种特异性的液滴数字PCR（ddPCR）分析方法。结果表明，该检测方法可将小肠芽孢杆菌与敦煌芽孢杆菌区分开，并导致约10个基因拷贝的检出限。此外，这些物种的ddPCR可用于从实验感染的仓鼠血液中提取的DNA，通过显微镜检查可检测出阴性的低寄生虫感染。总而言之，我们已经开发了灵敏的和特异性的定量ddPCR检测方法，用于检测血液中的B. microti和D. duncani。我们的方法可用作监测啮齿动物感染模型中寄生虫病进展的敏感方法，并可用作血液筛查中的合适分子检测。

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