This report describes the development, optimization, and validation of a ddPCR assay for the detection of Bartonella spp. DNA within several sample matrices, including clinical blood samples from patients with or without documented Bartonella spp. bacteremia. The Bartonella spp. ddPCR assay was developed based upon previously published TaqMan-based qPCR assays that can amplify DNA of over 25 Bartonella spp. Host DNA (housekeeping gene) amplification serves as a reference target to facilitate quantification. The efficiency, sensitivity, and specificity of the Bartonella spp. ddPCR assay was assessed by direct comparison with the current qPCR methods used by the Intracellular Pathogens Research Laboratory (North Carolina State University, North Carolina, USA), and Galaxy Diagnostics (Research Triangle Park, North Carolina, USA). Bartonella spp. ddPCR assay parameters were successfully optimized to detect Bartonella concentrations equivalent to 0.5 bacterial genome copies per microliter of blood (0.001 pg/ul of bacterial DNA). The number of droplets detected (resolution) for each concentration was consistent across each of four assessed time points. The Bartonella spp. ddPCR assay detected 16 species/strains including B. henselae; B. quintana; B. vinsonii subsp. berkhoffii (genotypes I, II, III and IV); B. vinsonii subsp. vinsonii; B. melophagi; B. volans; B. monaki; B. alsatica; B. bovis; B. elizabethae; B. clarridgeiae; and B. koehlerae. Bartonella DNA was detected in only one previously negative patient sample (119/120 negative; 99% specificity). The ddPCR sensitivity (53/112) was significantly better than qPCR (6/112) when testing patient blood and enrichment blood culture samples. The development of commercial ddPCR systems with integrated technologies has significantly streamlined the DNA detection process, making it more efficient and standardized for clinical diagnostic testing. The assay described in this work is the first step toward the development of a multiplex ddPCR assay (i.e., using the QX One from Bio-Rad) for the simultaneous detection and absolute quantification of multiple vector-borne pathogens (such as Babesia, Bartonella and Borrelia) within clinical samples.

本报告介绍了用于检测Bartonella spp的ddPCR检测方法的开发，优化和验证。几种样品基质中的DNA，包括有或没有有Bartonella spp的患者的临床血液样品。菌血症。在巴尔通体属。ddPCR测定法是基于以前发布的基于TaqMan的qPCR测定法开发的，可以扩增超过25个Bartonella spp的DNA 。宿主DNA（管家基因）扩增用作参考目标，有助于定量。巴尔通体的效率，敏感性和特异性spp。通过与细胞内病原体研究实验室（美国北卡罗来纳州北卡罗来纳州立大学）和银河系诊断公司（美国北卡罗莱纳州三角研究公园）目前使用的qPCR方法进行直接比较来评估ddPCR分析。巴尔通体属 ddPCR分析参数已成功优化，以检测相当于每微升血液0.5个细菌基因组拷贝（0.001 pg / ul细菌DNA）的巴尔通体浓度。在四个评估的时间点中的每个时间点，每种浓度检测到的液滴数量（分辨率）均保持一致。在巴尔通体属。ddPCR分析检测到16种/菌株，包括亨氏芽孢杆菌; B.通体; 温氏芽孢杆菌亚种 berkhoffii（基因型I，II，III和IV）；B.vinsonii亚种 vinsonii; 乙lo ; 苍蝇; B. monaki ; B. alsatica; 乙。牛头肌; B. elizabethae ; 克拉氏杆菌; 和koehlerae。巴通体仅在一个先前阴性的患者样本中检测到DNA（119/120阴性； 99％的特异性）。在测试患者血液和富集血液培养样本时，ddPCR灵敏度（53/112）明显优于qPCR（6/112）。具有集成技术的商业ddPCR系统的开发已大大简化了DNA检测过程，使其更加有效和标准化，可用于临床诊断测试。这项工作中描述的测定方法是朝着同时检测和绝对定量多种载体传播病原体（例如巴贝斯虫，巴尔通体和巴氏杆菌）的多重ddPCR测定法（即使用Bio-Rad的QX One）迈出的第一步。临床样品中的疏螺旋体）。