Department of Molecular Biosciences & Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
Department of Human Nutrition, Food, and Animal Science, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
PLoS One. 2021 Jan 5;16(1):e0244956. doi: 10.1371/journal.pone.0244956. eCollection 2021.
Constraints related to sample preparation are some of the primary obstacles to widespread deployment of molecular diagnostics for rapid detection of trace quantities (≤103 CFU/mL) of food-borne pathogens. In this research, we report a sample preparation method using a novel handheld electroflotation system to concentrate and recover dilute quantities (102-103 CFU/mL) of Escherichia coli (E. coli) 25922 in artificially contaminated samples for reliable, rapid detection by loop-mediated isothermal amplification (LAMP). To protect suspended cells from shear stresses at bubble surfaces, a non-ionic surfactant (Pluronic-F68) and flocculant (chitosan oligosaccharide) were used to aggregate cells and reduce their surface hydrophobicity. Effective conditions for recovery were determined through multifactorial experiments including various concentrations of Pluronic-F68 (0.001, 0.01, 0.1, 1 g L-1), chitosan oligosaccharide (0.01, 0.1, 1, 10 g L-1), bacteria (102, 103, 104 CFU/mL E. coli 25922), recovery times (10, 15 and 20 minutes), and degrees of turbulent gas flux ("high" and "low"). The automated electroflotation system was capable of concentrating effectively all of the bacteria from a large sample (380 mL 0.1 M potassium phosphate buffer containing 102 CFU/mL E. coli) into a 1 mL recovered fraction in less than 30 minutes. This enabled detection of bacterial contaminants within 2 hours of collecting the sample, without a specialized laboratory facility or traditional enrichment methods, with at least a 2-3 order of magnitude improvement in detection limit compared to direct assay with LAMP.
样品制备相关的限制因素是广泛部署分子诊断技术用于快速检测痕量（≤103 CFU/mL）食源性病原体的主要障碍之一。在本研究中，我们报告了一种使用新型手持式电浮选系统浓缩和回收人工污染样品中稀释数量（102-103 CFU/mL）的大肠杆菌（E. coli）25922 的样品制备方法，用于可靠、快速的环介导等温扩增（LAMP）检测。为了保护悬浮细胞免受气泡表面的剪切力影响，使用非离子表面活性剂（Pluronic-F68）和絮凝剂（壳聚糖寡糖）来聚集细胞并降低其表面疏水性。通过包括各种 Pluronic-F68 浓度（0.001、0.01、0.1、1 g L-1）、壳聚糖寡糖浓度（0.01、0.1、1、10 g L-1）、细菌数量（102、103、104 CFU/mL E. coli 25922）、回收时间（10、15 和 20 分钟）和气体通量强度（“高”和“低”）的多因素实验确定了有效的回收条件。自动化电浮选系统能够在不到 30 分钟的时间内将大量样品（380 mL 0.1 M 磷酸钾缓冲液，含 102 CFU/mL E. coli）中的所有细菌有效地浓缩到 1 mL 回收部分中。这使得在采集样品后 2 小时内即可检测到细菌污染物，无需专门的实验室设施或传统的富集方法，与直接进行 LAMP 检测相比，检测限至少提高了 2-3 个数量级。
