A pipette-actuated capillary array comb (PAAC) system operated on a smartphone-based hand-held device has been successfully developed for the multiplex detection of bacteria in a “sample-to-answer” manner. The PAAC consists of eight open capillaries inserted into a cylindrical plastic base with a piece of chitosan-modified glass filter paper embedded in each capillary. During the sample preparation, a PAAC was mounted into a 1 mL pipette tip with an enlarged opening and was operated with a 1 mL pipette for liquid handling. The cell lysate was drawn and expelled through the capillaries three times to facilitate the DNA capture on the embedded filter discs. Following washes with water, the loop-mediated isothermal amplification (LAMP) reagents were aspirated into the capillaries, in which the primers were pre-fixed with chitosan. After that, the PAAC was loaded into the smartphone-based device for a one-hour amplification at 65 °C and end-point detection of calcein fluorescence in the capillaries. The DNA capture efficiency of a 1.1 mm-diameter filter disc was determined to be 97% of λ-DNA and the coefficient of variation among the eight capillaries in the PAAC was only 2.2%. The multiplex detection of genomic DNA extracted from Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus provided limits of detection of 200, 500, and 500 copies, respectively, without any cross-contamination and cross reactions. “Sample-to-answer” detection of E. coli samples was successfully completed in 85 minutes, demonstrating a sensitivity of 200 cfu per capillary. The multiplex “sample-to-answer” detection, the streamlined operation, and the compact device should facilitate a broad range of applications of our PAAC system in point-of-care testing.

中文翻译：

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使用带有集成DNA提取，等温扩增和智能手机检测功能的移液器驱动毛细管阵列梳进行细菌的从样品到答案的多重检测†

在基于智能手机的手持设备上操作的移液器驱动的毛细管阵列梳（PAAC）系统已成功开发，可以“样品到答案”的方式对细菌进行多重检测。PAAC由八个开放式毛细管组成，这些毛细管插入圆柱形塑料基座中，每个毛细管中均嵌有一块经脱乙酰壳多糖改性的玻璃滤纸。在样品制备过程中，将PAAC安装到具有扩大开口的1 mL移液器吸头中，并用1 mL移液器进行操作以进行液体处理。提取细胞裂解物，并通过毛细血管排出三遍，以促进DNA在嵌入式滤盘上的捕获。用水洗涤后，将环介导的等温扩增（LAMP）试剂吸入毛细管中，在其中将引物预先用壳聚糖固定。之后，将PAAC加载到基于智能手机的设备中，在65°C下进行一小时的扩增，并在毛细管中检测钙黄绿素荧光的终点。经测定，直径为1.1毫米的滤盘的DNA捕获效率为λ-DNA的97％，而PAAC中八个毛细管之间的变异系数仅为2.2％。从中提取的基因组DNA的多重检测大肠杆菌，肺炎克雷伯菌和金黄色葡萄球菌分别提供200、500和500拷贝的检出限，没有任何交叉污染和交叉反应。在85分钟内成功完成了对大肠杆菌样品的“从样品到答案”的检测，表明每个毛细管的灵敏度为200 cfu。多重“从样品到答案”的检测，简化的操作以及紧凑的设备，应有助于我们的PAAC系统在即时检验中的广泛应用。