Maggi Ricardo G, Richardson Toni, Breitschwerdt Edward B, Miller Jennifer C
Galaxy Diagnostics, Inc, 7020 Kit Creek Rd #130, Research Triangle Park, NC 27709, USA; Department of Clinical Sciences, The Comparative Medicine Institute, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC 27607, USA.
Galaxy Diagnostics, Inc, 7020 Kit Creek Rd #130, Research Triangle Park, NC 27709, USA.
J Microbiol Methods. 2020 Sep;176:106022. doi: 10.1016/j.mimet.2020.106022. Epub 2020 Aug 11.
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.
本报告描述了一种用于检测多种样本基质中巴尔通体属DNA的数字滴式PCR（ddPCR）检测方法的开发、优化和验证，这些样本基质包括有或无巴尔通体属菌血症记录的患者的临床血液样本。巴尔通体属ddPCR检测方法是基于先前发表的基于TaqMan的定量PCR（qPCR）检测方法开发的，该方法可扩增超过25种巴尔通体属的DNA。宿主DNA（管家基因）扩增用作参考靶点以促进定量。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Galaxy诊断公司（美国北卡罗来纳州三角研究园）目前使用的qPCR方法直接比较，评估了巴尔通体属ddPCR检测方法的效率、灵敏度和特异性。成功优化了巴尔通体属ddPCR检测方法的参数，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。在四个评估时间点的每一个中，每个浓度检测到的液滴数量（分辨率）都是一致的。巴尔通体属ddPCR检测方法检测到16种/菌株，包括亨氏巴尔通体；五日热巴尔通体；文森巴尔通体伯克霍夫亚种（基因型I、II、III和IV）；文森巴尔通体文森亚种；嗜蜱巴尔通体；飞鼠巴尔通体；莫纳基巴尔通体；阿尔萨蒂亚巴尔通体；牛巴尔通体；伊丽莎白巴尔通体；克拉里奇巴尔通体；以及科赫勒巴尔通体。仅在一份先前为阴性的患者样本中检测到巴尔通体DNA（120份样本中119份为阴性；特异性为99%）。在检测患者血液和富集血培养样本时，ddPCR的灵敏度（53/112）显著优于qPCR（6/112）。集成技术的商业ddPCR系统的开发显著简化了DNA检测过程，使其在临床诊断检测中更高效、更标准化。本研究中描述的检测方法是朝着开发用于同时检测和绝对定量临床样本中多种媒介传播病原体（如巴贝斯虫、巴尔通体和疏螺旋体）的多重ddPCR检测方法（即使用伯乐公司的QX One）迈出的第一步。
