Shen Zhenyu, Zhang Michael Z, Stich Roger W, Mitchell William J, Zhang Shuping
Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
J Microbiol Methods. 2018 Aug;151:83-89. doi: 10.1016/j.mimet.2018.05.019. Epub 2018 May 23.
Anaplasma spp., Ehrlichia spp., Rickettsia spp., and Lyme disease associated Borrelia spp. are the most common tick-borne pathogens reported to infect human beings worldwide and other animals, such as dogs and horses. In the present study, we developed a broad-coverage SYBR Green QPCR panel consisting of four individual assays for the detection and partial differentiation of the aforementioned pathogens. All assays were optimized to the same thermocycling condition and had a detection limit of 10 copies per reaction. The assays remained sensitive when used to test canine and equine blood DNA samples spiked with known amounts of synthetic DNA (gBlock) control template. The assays were specific, as evidenced by lack of cross reaction to non-target gBlock or other pathogens commonly tested in veterinary diagnostic labs. With appropriate Ct cutoff values for positive samples and negative controls and the melting temperature (TM) ranges established in the present study, the QPCR panel is suitable for accurate, convenient and rapid screening and confirmation of tick-borne pathogens in animals.
无形体属、埃立克体属、立克次体属以及与莱姆病相关的疏螺旋体属是全球范围内报道的最常见的可感染人类及其他动物（如犬类和马匹）的蜱传病原体。在本研究中，我们开发了一种覆盖范围广泛的SYBR Green QPCR检测板，它由四个单独的检测方法组成，用于检测和部分区分上述病原体。所有检测方法均针对相同的热循环条件进行了优化，每个反应的检测限为10个拷贝。当用于检测添加了已知量合成DNA（gBlock）对照模板的犬类和马类血液DNA样本时，这些检测方法仍保持灵敏。这些检测方法具有特异性，对非靶标gBlock或兽医诊断实验室中通常检测的其他病原体无交叉反应即可证明。通过本研究确定的阳性样本和阴性对照的适当Ct截止值以及熔解温度（TM）范围，该QPCR检测板适用于准确、便捷且快速地筛查和确认动物中的蜱传病原体。
