Huadong Research Institute for Medicine and Biotechniques, Nanjing, Jiangsu, China.
China Pharmaceutical University, Nanjing, Jiangsu, China.
PLoS One. 2018 Nov 26;13(11):e0207811. doi: 10.1371/journal.pone.0207811. eCollection 2018.
Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever, which is the most severe spotted fever group (SFG) rickettsiosis. Developing a simple and reliable detection method is required.
A detection method for R. rickettsii was established based on a recombinase polymerase amplification (RPA) assay and the lateral flow (LF) test. A specific target sequence was screened, and corresponding primers and probes were designed, synthesized, and screened for establishing an RPA assay with high amplification efficiency. Reagent concentrations, amplification time, and loading volume for strip development were optimized. The detection limit, analytic sensitivity and specificity were evaluated.
A rapid, visual, sensitive and specific method for the detection of R. rickettsii based on RPA and the LF test was successfully established. The novel method had a limit of detection of 10 to 50 copies/reaction without recognizing other organisms. Analytical sensitivity and specificity were ≥90% and 100%, respectively, as evaluated by animal and simulative human samples.
Using the established method, detection could be completed in 30 min with visually detectable results by the naked eye, without requirement of any instrument except a constant temperature equipment. The technique shows superior detection performance and is promising for wide use in the field as well as resource-limited areas for R. rickettsii detection.
落矶山斑点热是由斑点热群立克次体（SFG）中的恙虫病东方体引起的，是最严重的斑点热群疾病。因此，需要开发一种简单可靠的检测方法。
本研究基于重组酶聚合酶扩增（RPA）检测和侧向流动（LF）检测，建立了一种恙虫病东方体检测方法。筛选出一个特定的目标序列，设计、合成相应的引物和探针，并进行筛选，以建立具有高扩增效率的 RPA 检测。优化了条带开发的试剂浓度、扩增时间和加载体积。评估了检测限、分析灵敏度和特异性。
成功建立了一种基于 RPA 和 LF 检测的快速、直观、敏感和特异的恙虫病东方体检测方法。该新方法的检测限为 10 至 50 拷贝/反应，并且不会识别其他生物体。通过动物和模拟人体样本评估，分析灵敏度和特异性分别≥90%和 100%。
使用所建立的方法，在 30 分钟内即可完成肉眼可见的检测，除了恒温设备外，不需要任何仪器。该技术具有优越的检测性能，有望在现场和资源有限的地区广泛用于恙虫病东方体的检测。
