Pathogen Concentration Combined Solid-Phase PCR on Supercritical Angle Fluorescence Microlens Array for Multiplexed Detection of Invasive Nontyphoidal Salmonella Serovars.

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.