Ngamsom Bongkot, Lopez-Martinez Maria J, Raymond Jean-Claude, Broyer Patrick, Patel Pradip, Pamme Nicole
Department of Chemistry, University of Hull, Hull HU6 7RX, UK.
bioMérieux Innovation, Chemin de I'Orme, Marcy I'Etoile, 69280, France.
J Microbiol Methods. 2016 Apr;123:79-86. doi: 10.1016/j.mimet.2016.01.016. Epub 2016 Feb 4.
Pathogen analysis in food samples routinely involves lengthy growth-based pre-enrichment and selective enrichment of food matrices to increase the ratio of pathogen to background flora. Similarly, for blood culture analysis, pathogens must be isolated and enriched from a large excess of blood cells to allow further analysis. Conventional techniques of centrifugation and filtration are cumbersome, suffer from low sample throughput, are not readily amenable to automation and carry a risk of damaging biological samples. We report on-chip acoustophoresis as a pre-analytical technique for the resolution of total microbial flora from food and blood samples. The resulting 'clarified' sample is expected to increase the performance of downstream systems for the specific detection of the pathogens. A microfluidic chip with three inlets, a central separation channel and three outlets was utilized. Samples were introduced through the side inlets, and buffer solution through the central inlet. Upon ultrasound actuation, large debris particles (10-100 μm) from meat samples were continuously partitioned into the central buffer channel, leaving the 'clarified' outer sample streams containing both, the pathogenic cells and the background flora (ca. 1 μm) to be collected over a 30 min operation cycle before further analysis. The system was successfully tested with Salmonella typhimurium-spiked (ca. 10(3)CFU mL(-1)) samples of chicken and minced beef, demonstrating a high level of the pathogen recovery (60-90%). When applied to S. typhimurium contaminated blood samples (10(7)CFU mL(-1)), acoustophoresis resulted in a high depletion (99.8%) of the red blood cells (RBC) which partitioned in the buffer stream, whilst sufficient numbers of the viable S. typhimurium remained in the outer channels for further analysis. These results indicate that the technology may provide a generic approach for pre-analytical sample preparation prior to integrated and automated downstream detection of bacterial pathogens.
食品样本中的病原体分析通常需要基于生长的长时间预富集以及对食品基质进行选择性富集，以提高病原体与背景菌群的比例。同样，对于血培养分析，必须从大量多余的血细胞中分离并富集病原体，以便进行进一步分析。传统的离心和过滤技术操作繁琐，样本通量低，不易实现自动化，并且存在损坏生物样本的风险。我们报道了芯片上的声泳技术作为一种预分析技术，用于从食品和血液样本中分离总微生物菌群。预期由此得到的“澄清”样本将提高下游系统对病原体进行特异性检测的性能。使用了一种具有三个入口、一个中央分离通道和三个出口的微流控芯片。样本通过侧面入口引入，缓冲溶液通过中央入口引入。在超声驱动下，肉类样本中的大碎片颗粒（10 - 100μm）持续被分隔到中央缓冲通道中，在30分钟的操作周期内收集包含致病细胞和背景菌群（约1μm）的“澄清”外部样本流，然后进行进一步分析。该系统成功地用添加了鼠伤寒沙门氏菌（约10³CFU mL⁻¹）的鸡肉和牛肉末样本进行了测试，病原体回收率很高（60 - 90%）。当应用于被鼠伤寒沙门氏菌污染的血液样本（10⁷CFU mL⁻¹）时，声泳导致在缓冲流中分隔的红细胞（RBC）高度耗尽（99.8%），而足够数量的存活鼠伤寒沙门氏菌留在外部通道中以便进一步分析。这些结果表明，该技术可能为在对细菌病原体进行集成和自动化下游检测之前的预分析样本制备提供一种通用方法。
