AN IMPROVED PCR PROTOCOL FOR DETECTION OF BABESIA DUNCANI IN WILDLIFE AND VECTOR SAMPLES
AN IMPROVED PCR PROTOCOL FOR DETECTION OF BABESIA DUNCANI IN WILDLIFE AND VECTOR SAMPLES
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DOI：
10.1645/17-155
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发表时间：
2018-08-01
影响因子：
1.3
通讯作者：
Swei, A.
中科院分区：
文献类型：
--
作者：
O'Connor, K. E.;Kjemtrup, A. M.;Swei, A.
人巴贝斯虫病是一种由硬蜱传播的原生动物疾病，在北美具有越来越重要的临床意义。据报道，美国东部和中西部地区的大多数病例都是由微小巴贝斯虫感染引起的。相比之下，加利福尼亚州和华盛顿州报告的大多数人类感染被归因于一种新物种，该物种于1991年首次被发现，随后被命名为邓肯巴贝西亚。虽然对微小白纹伊蚊的媒介媒介和哺乳动物宿主有很好的描述，但对邓肯白纹伊蚊的媒介和宿主尚不清楚。因此，无法确定人类感染的具体风险因素。由于缺乏特异和灵敏的分子诊断工具来扩增寄生虫DNA，对潜在宿主和媒介物种的识别一直受到阻碍。为了满足这一需要，建立了针对β-微管蛋白基因的巢式聚合酶链式反应方法，β-微管蛋白基因是一种在不同物种之间具有高度可变内含子区域的锥体寄生虫中保守的基因。用来自人类患者(WA-1)的邓肯分支杆菌分离株(WA-1)的DNA和相关的巴贝斯虫种的DNA，通过添加扁虱和哺乳动物DNA提取液来评估该方法。来自加州的野生动物。该方法具有很高的特异性，其灵敏度约为1个拷贝的模板DNA。在这种水平的检测中，在幼虫样本中可以检测到邓肯芽胞杆菌，并且目标位置允许通过凝胶电泳在物种之间进行视觉区分。本实验为研究人员阐明邓肯分枝杆菌的自然传播周期提供了新的工具。
Human babesiosis is a tick-borne protozoal disease of increasing clinical significance in North America. Most cases in the eastern and Midwestern regions of the United States are reportedly due to Babesia microti infections. By contrast, most human infections reported in California and Washington have been attributed to a new species that was first identified in 1991 and subsequently named Babesia duncani. Although the tick vector and mammalian reservoir hosts for B. microti are well characterized, the vector and reservoir hosts for B. duncani are unknown. As a result, specific risk factors for human infections cannot be characterized. Identification of potential hosts and vector species has been hampered by the lack of specific and sensitive molecular diagnostic tools to amplify parasite DNA. To address this need, a nested PCR assay targeting the beta-tubulin gene, a well-conserved locus in piroplasm parasites with a highly variable intron region among species, was developed. The assay was evaluated by spiking tick and mammalian DNA extracts with DNA from a B. duncani isolate derived from a human patient (WA-1) as well as related Babesia spp. from Californian wildlife. This assay was highly specific, with a sensitivity of approximately 1 copy of template DNA in a background of tick DNA. At this level of detection B. duncani was detectable in larval tick samples, and the target locus allowed for visual differentiation between species by gel electrophoresis. This assay offers researchers a new tool for elucidating the natural transmission cycle of B. duncani.