Graham Christine B, Pilgard Mark A, Maes Sarah E, Hojgaard Andrias, Eisen Rebecca J
Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, 3156 Rampart Rd., Fort Collins, CO 80521, United States.
Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, 3156 Rampart Rd., Fort Collins, CO 80521, United States.
Ticks Tick Borne Dis. 2016 Oct;7(6):1230-1235. doi: 10.1016/j.ttbdis.2016.07.009. Epub 2016 Jul 18.
Borrelia miyamotoi is an emerging, tick-borne human pathogen. In North America, it is primarily associated with Ixodes scapularis and Ixodes pacificus, two species known to bite humans. Here we describe the development and evaluation of a pair of real-time TaqMan PCR assays designed to detect B. miyamotoi in North American ticks. We sought to achieve sensitivity to B. miyamotoi strains associated with ticks throughout North America, the full genetic diversity of which is unknown, by targeting sequences that are largely conserved between B. miyamotoi strains from the eastern United States and genetically distinct B. miyamotoi strains from Japan. The two assays target different loci on the B. miyamotoi chromosome and can be run side by side under identical cycling conditions. One of the assays also includes a tick DNA target that can be used to verify the integrity of tick-derived samples. Using both recombinant plasmid controls and genomic DNA from North American and Japanese strains, we determined that both assays reliably detect as few as 5 copies of the B. miyamotoi genome. We verified that neither detects B. burgdorferi, B. lonestari or B. turicatae. This sensitive and specific pair of assays successfully detected B. miyamotoi in naturally-infected, colony-reared nymphs and in field-collected I. scapularis and I. pacificus from the Northeast and the Pacific Northwest respectively. These assays will be useful in screening field-collected Ixodes spp. from varied regions of North America to assess the risk of human exposure to this emerging pathogen.
宫本疏螺旋体是一种新出现的、通过蜱传播的人类病原体。在北美，它主要与肩突硬蜱和太平洋硬蜱有关，这两种蜱都已知会叮咬人类。在此，我们描述了一对实时TaqMan PCR检测方法的开发和评估，旨在检测北美蜱中的宫本疏螺旋体。我们试图通过靶向美国东部宫本疏螺旋体菌株与来自日本的基因不同的宫本疏螺旋体菌株之间基本保守的序列，来实现对与北美各地蜱相关的宫本疏螺旋体菌株的敏感性，其完整的遗传多样性尚不清楚。这两种检测方法针对宫本疏螺旋体染色体上的不同位点，可在相同的循环条件下并行运行。其中一种检测方法还包括一个蜱DNA靶点，可用于验证蜱源样本的完整性。使用重组质粒对照以及来自北美和日本菌株的基因组DNA，我们确定这两种检测方法都能可靠地检测到低至5个拷贝的宫本疏螺旋体基因组。我们验证了这两种方法均未检测到伯氏疏螺旋体、孤独疏螺旋体或图莱里疏螺旋体。这一对灵敏且特异的检测方法成功地在自然感染的、群体饲养的若虫以及分别从美国东北部和太平洋西北部野外采集的肩突硬蜱和太平洋硬蜱中检测到了宫本疏螺旋体。这些检测方法将有助于筛选从北美不同地区野外采集的硬蜱属蜱虫，以评估人类接触这种新出现病原体的风险。
