Bloodstream infections and invasive nontyphoidal Salmonellosis in particular remain a major health and economic burden worldwide. The complexity of blood matrixes along with extremely low concentration of pathogens in blood poses a great challenge for rapid and ultrasensitive detection. Sample preparation has been the critical step that should provide blood-matrix-free sample with the targeted pathogen in the highest possible concentration. In this work, we addressed this challenge by combining magnetic-bead-based pathogen concentration and solid-phase PCR (SP-PCR). The SP-PCR performed on a supercritical angle fluorescence (SAF) microlens array embedded in a microchip enabled quick and accurate detection of low levels of Salmonella enterica serovar typhimurium and enteritidis in blood samples without culture enrichment. Protein AG-magnetic beads immobilized with antisalmonella antibody could efficiently concentrate both Salmonella serovars with a capturing efficiency >95%. Higher tolerance of Phusion hot start DNA polymerase to PCR inhibitors and its compatibility with protein AG-magnetic beads allowed the integration of SP-PCR. Analysis of Salmonella-spiked blood samples with the SP-PCR resulted in a limit of detection (LoD) as low as 86 CFU/mL and 94 CFU/mL for S. typhimurium and S. enteritidis, respectively, that could be attributed to the high fluorescence collection efficiency of the SAF microlens array. These combinations reduced the duration of analysis to less than 3 h including sample preparation. This platform has the potential for wide application as a high-throughput biosensor to analyze pathogens in clinical, food, and environmental samples.

中文翻译：

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超临界角荧光微透镜阵列上的病原体浓缩结合固相PCR用于入侵性非伤寒沙门氏菌血清的多重检测。

尤其是血流感染和侵入性非伤寒沙门氏菌病仍然是世界范围内的主要健康和经济负担。血液基质的复杂性以及血液中病原体的浓度极低，对快速，超灵敏的检测提出了巨大挑战。样品制备一直是至关重要的步骤，应以尽可能高的浓度向无血基质样品提供目标病原体。在这项工作中，我们通过结合基于磁珠的病原体浓度和固相PCR（SP-PCR）解决了这一挑战。在微芯片中嵌入的超临界角荧光（SAF）微透镜阵列上进行的SP-PCR能够快速准确地检测血液样品中低水平的肠炎沙门氏菌血清鼠伤寒沙门氏菌和肠炎沙门氏菌，而无需进行培养物富集。固定有抗沙门氏菌抗体的蛋白AG磁珠可以有效地浓缩两种沙门氏菌，捕获效率> 95％。Phusion热启动DNA聚合酶对PCR抑制剂的更高耐受性及其与蛋白质AG磁珠的相容性使得SP-PCR可以整合。用SP-PCR分析沙门氏菌加标血样的结果，鼠伤寒沙门氏菌和肠炎沙门氏菌的检出限（LoD）分别低至86 CFU / mL和94 CFU / mL。 SAF微透镜阵列的高荧光收集效率。这些组合将包括样品前处理在内的分析时间缩短至不到3小时。该平台具有作为高通量生物传感器广泛应用的潜力，可用于分析临床，食品和环境样品中的病原体。