College of Veterinary Medicine, Western University of Health Sciences, Pomona, California, USA.
Microbiol Spectr. 2023 Jun 15;11(3):e0439522. doi: 10.1128/spectrum.04395-22. Epub 2023 May 11.
Infectious disease diagnostics often depend on costly serological testing with poor sensitivity, low specificity, and long turnaround time. Here, we demonstrate proof of the principle for simultaneous detection of two tick-borne pathogens from a single test sample using barcoded magnetic bead technology on the BioCode 2500 system. Specific primer sets complementary to the conserved genes of Anaplasma phagocytophilum and Borrelia burgdorferi were used in PCR amplification of the target, followed by the hybridization of the resulting biotinylated PCR products with specific probes tethered to the barcoded magnetic beads for simultaneous detection, using a fluorophore with high quantum yield. The assay has an extremely high signal to background ratio, with a limit of detection (LOD) of 2.81 50% tissue culture infection dose (TCID)/mL and 1 CFU/mL for A. phagocytophilum and B. burgdorferi, respectively. The observed LOD for gene blocks was 1.8 copies/reaction for both the pathogens. The assay demonstrated 100% positive and negative agreement on performance evaluation using patient specimens and blood samples spiked with 1 × LOD of pathogen stock. No cross-reactivity was observed with other related tick-borne pathogens and genomic DNA of human, cattle, and canine origin. The assay can be upgraded to a sensitive and cost-effective multiplex diagnostic approach that can simultaneously detect multiple clinically important tick-borne pathogens in a single sample with a short turnaround time. The low pathogen load in the tick-borne disease test samples and the lack of highly sensitive multiplex diagnostic approaches have impacted diagnosis during clinical testing and limited surveillance studies to gauge prior insight about the prevalence of tick-borne infections in a geographical area. This article demonstrates proof of the principle for simultaneous detection of two important tick-borne pathogens from a single test sample using digital barcoded magnetic bead technology. Using a fluorophore of high quantum yield, the diagnostic approach showed high sensitivity and specificity. The LOD was 1.8 genome copies per reaction for both A. phagocytophilum and B. burgdorferi. The assay can be upgraded for the detection of all clinically important tick-borne pathogens from a single patient sample with high sensitivity and specificity. The assay can provide a diagnostic answer to the clinician in a short turnaround time to facilitate speedy therapeutic intervention to infected patients and implement public health measures to prevent community spread.
传染病诊断通常依赖于昂贵的血清学检测，但这种检测方法的灵敏度低、特异性差，且周转时间长。在这里，我们使用条码磁珠技术在 BioCode 2500 系统上展示了从单个测试样本中同时检测两种蜱传病原体的原理验证。针对靶基因的保守序列，我们使用了互补的特异性引物进行 PCR 扩增，然后将生物素化的 PCR 产物与特定探针杂交，这些探针通过条码磁珠固定，同时检测，使用高荧光量子产率的荧光染料。该检测方法具有极高的信号与背景比值，其检测限（LOD）分别为 2.81 50%组织培养感染剂量（TCID）/mL 和 1 CFU/mL，用于检测嗜吞噬细胞无形体和伯氏疏螺旋体。对于基因块，检测限分别为两种病原体的 1.8 个拷贝/反应。该检测方法在使用患者标本和用病原体库存 1×LOD 稀释的血液样本进行性能评估时，显示出 100%的阳性和阴性符合率。该检测方法与其他相关蜱传病原体以及人、牛和犬的基因组 DNA 无交叉反应性。该检测方法可以升级为一种灵敏且经济有效的多重诊断方法，可在短时间内从单个样本中同时检测多种临床上重要的蜱传病原体。由于蜱传疾病测试样本中的病原体载量低，且缺乏高灵敏度的多重诊断方法，这影响了临床检测中的诊断，限制了监测研究，无法全面了解该地区蜱传感染的流行情况。本文展示了使用数字条码磁珠技术从单个测试样本中同时检测两种重要蜱传病原体的原理验证。该诊断方法使用高荧光量子产率的荧光染料，具有高灵敏度和特异性。对于嗜吞噬细胞无形体和伯氏疏螺旋体，检测限分别为 1.8 个基因组拷贝/反应。该检测方法可以升级为用于从单个患者样本中同时检测所有临床上重要的蜱传病原体，具有高灵敏度和特异性。该检测方法可以在短时间内向临床医生提供诊断结果，以便为感染患者提供快速治疗干预，并采取公共卫生措施来防止社区传播。
