Early and accurate detection of pathogens is important to improve clinical outcomes of bloodstream infections (BSI), especially in the case of drug-resistant pathogens. In this study, we aimed to develop a culture-independent digital PCR (dPCR) system for multiplex detection of major sepsis-causing gram-negative pathogens and antimicrobial resistance genes using plasma DNA from BSI patients. Our duplex dPCR system successfully detected nine targets (five bacteria-specific targets and four antimicrobial resistance genes) through five reactions within 3 hours. The minimum detection limit was 50 ag of bacterial DNA, suggesting that 1 CFU/ml of bacteria in the blood can be detected. To validate the clinical applicability, cell-free DNA samples from febrile patients were tested with our system and confirmed high consistency with conventional blood culture. This system can support early identification of some drug-resistant gram-negative pathogens, which can help improving treatment outcomes of BSI.

中文翻译：

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用于快速识别血流感染患者血液中革兰氏阴性病原体的双重 dPCR 系统：一种不依赖于培养的方法。

病原体的早期和准确检测对于改善血流感染 (BSI) 的临床结果非常重要，尤其是在耐药病原体的情况下。在这项研究中，我们旨在开发一种不依赖于培养物的数字 PCR (dPCR) 系统，用于使用 BSI 患者的血浆 DNA 对主要引起败血症的革兰氏阴性病原体和抗菌素耐药基因进行多重检测。我们的双重 dPCR 系统在 3 小时内通过五个反应成功检测到九个目标（五个细菌特异性目标和四个抗菌素耐药基因）。最低检测限为 50 μg 的细菌 DNA，表明可以检测到血液中 1 CFU/ml 的细菌。为了验证临床适用性，来自发热患者的游离 DNA 样本用我们的系统进行了测试，并证实与传统血培养具有高度一致性。该系统可以支持早期识别一些耐药的革兰氏阴性病原体，这有助于改善 BSI 的治疗结果。