Medicity Research Laboratories and Laboratory of Biophysics, Institute of Biomedicine, Faculty of Medicine, University of Turku, Finland.
Clinical Microbiology Laboratory, Turku University Hospital and University of Turku, Finland.
Anal Biochem. 2019 Apr 1;570:21-26. doi: 10.1016/j.ab.2019.01.011. Epub 2019 Feb 5.
Urinary tract infections (UTIs) are a common problem worldwide. The most prevalent causative pathogen of UTI is Escherichia coli, focus of this study. The current golden standard for detecting UTI is bacterial culture, creating a major workload for hospital laboratories - cost-effective and rapid mass screening of patient samples is needed. Here we present an alternative approach to screen patient samples with a single-step assay utilising time-resolved luminescence and luminescence modulating biosensing phages. Filamentous phage M13 was biopanned for binding luminescence quenching metal (copper) and further E. coli. The screening assay luminescence modulation was further enhanced by selecting right chemical environment for the functioning phage clones. Semi-specific interaction between phage, target bacteria and metal was detected by modulation in the signal of a weakly chelating, easily quenchable lanthanide complex. In the presence of the target pathogen, the phages collected quenching metal from solution to the bacterial surface changing the quenching effect on the lanthanide label and thus modulating the signal. Our method was compared with the bacterial culture data obtained from 70 patient samples. The developed proof-of-principle screening assay showed sensitivity and a specificity at the 90% mark when compared to culture method although some samples had high turbidity and even blood. The detection limit of E. coli was in the range of 1000-10 000 colony forming units/mL. Untreated urine sample was screened and time-resolved luminescence signal result was achieved within 10 min in a single incubation step.
尿路感染（UTI）是全球范围内的常见问题。UTI 最常见的病原体是大肠杆菌，这是本研究的重点。目前检测 UTI 的金标准是细菌培养，这给医院实验室带来了很大的工作量——需要经济高效且快速的对患者样本进行大规模筛查。在这里，我们提出了一种替代方法，利用时间分辨发光和发光调节生物传感噬菌体的一步法检测来筛选患者样本。丝状噬菌体 M13 经过生物淘选，用于结合发光猝灭金属（铜）和进一步的大肠杆菌。通过为功能噬菌体克隆选择合适的化学环境，进一步增强了筛选检测的发光调制。噬菌体、靶细菌和金属之间的半特异性相互作用通过对弱螯合、易猝灭的镧系元素配合物信号的调制来检测。在存在靶病原体的情况下，噬菌体从溶液中收集猝灭金属到细菌表面，改变对镧系元素标记的猝灭效应，从而调节信号。我们的方法与从 70 个患者样本中获得的细菌培养数据进行了比较。尽管一些样本的浊度很高甚至有血液，但与培养方法相比，开发的原理验证筛选检测法的灵敏度和特异性达到了 90%。大肠杆菌的检测限在 1000-10000 个菌落形成单位/mL 范围内。在未经处理的尿液样本中进行了筛选，并在单个孵育步骤中在 10 分钟内获得了时间分辨发光信号结果。
