Ross Anne-Marie L, Stokes John V, Cross Claire E, Alugubelly Navatha, Varela-Stokes Andrea S
Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS 39762, USA.
Pathogens. 2022 May 18;11(5):594. doi: 10.3390/pathogens11050594.
Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a valuable biomedical model for studying Spotted Fever Rickettsiosis (SFR); its immune system is more like the human immune system than that of the murine model, and guinea pigs develop characteristic clinical signs. Thus, we have a compelling interest in developing, expanding, and optimizing tools for use in our guinea pig-- system for understanding host-tick-pathogen interactions. With the design and optimization of the three multiplex TaqMan qPCR assays described here, we can detect the two SFGR, their respective primary sp. vectors, and the guinea pig model as part of controlled experimental studies using tick-transmission of SFGR to guinea pigs. We developed qPCR assays that reliably detect each specific target down to 10 copies by producing plasmid standards for each assay target, optimizing the individual primer-probe sets, and optimizing the final multiplex reactions in a methodical, stepwise fashion. We anticipate that these assays, currently designed for in vivo studies, will serve as a foundation for optimal SFGR detection in other systems, including fieldwork.
斑点热立克次体病（SFR）由斑点热群立克次体（SFGR）引起，且与其他疾病的常见症状相关，这使得在检测到SFGR特异性抗体之前进行诊断具有挑战性。豚鼠是研究斑点热立克次体病（SFR）的一种有价值的生物医学模型；其免疫系统比小鼠模型更类似于人类免疫系统，并且豚鼠会出现特征性临床症状。因此，我们对开发、扩展和优化用于我们豚鼠系统的工具有着浓厚兴趣，以了解宿主-蜱-病原体之间的相互作用。通过本文所述的三种多重TaqMan qPCR检测方法的设计和优化，我们能够在使用蜱将SFGR传播给豚鼠的对照实验研究中，检测两种SFGR、它们各自的主要蜱传播媒介以及豚鼠模型。我们通过为每个检测靶点制备质粒标准品、优化各个引物-探针组，并以有条不紊、逐步推进的方式优化最终的多重反应，开发出了能够可靠地检测低至10个拷贝的每个特定靶点的qPCR检测方法。我们预计，这些目前为体内研究设计的检测方法，将为在包括野外工作在内的其他系统中进行最佳SFGR检测奠定基础。
