Rapid bacterial detection and antibiotic susceptibility testing in whole blood using one-step, high throughput blood digital PCR

Abram, Timothy J.; Cherukury, Hemanth; Ou, Chen-Yin; Vu, Tam; Toledano, Michael; Li, Yiyan; Grunwald, Jonathan T.; Toosky, Melody N; Tifrea, Delia F.; Slepenkin, Anatoly; Chong, Jonathan; Kong, Lingshun; Pozo, Domenica Vanessa Del; La, Kieu; Labanieh, Louai; Zimak, Jan; Shen, Byron; Huang, Susan S.; Gratton, Enrico; Peterson, Ellena M.; Zhao, Weian

doi:10.1039/c9lc01212e

Cited by 87 publications

(68 citation statements)

References 45 publications

(57 reference statements)

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“…48 Abram et al reported a one-step ddPCR assay for rapid detection of multiple antibiotic resistance genes directly from whole blood specimens, which can achieve a sensitivity of 10 CFU mL À1 with a rapid sample-to-answer assay time (1 hour). 50 However, for genotypic ASTs, detailed knowledge of which resistance genes to test is required, which makes the methods not feasible for detecting unknown antibiotic resistant species. Since new forms of resistance evolve quickly, predicting resistance by analyzing a few known resistance genes is not a general solution.…”

Section: Digital Pcr and Digital Lamp For Rapid Astsmentioning

confidence: 99%

Microfluidic systems for rapid antibiotic susceptibility tests (ASTs) at the single-cell level

Zhang

Qin

et al. 2020

Chem. Sci.

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Section: Digital Pcr and Digital Lamp For Rapid Astsmentioning

confidence: 99%

Microfluidic systems for rapid antibiotic susceptibility tests (ASTs) at the single-cell level

Zhang

Qin

et al. 2020

Chem. Sci.

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“…For the processing of monodisperse droplets, the existing microfluidic technologies can be classified into two general categories: (i) On-chip systems, where the droplets remain in the cartridge and analysis and droplet manipulation are performed on-chip [9][10][11][12][13][14][15][16]. (ii) Off-chip systems, where the droplets are transferred into a reaction tube and downstream analysis and/or further manipulation steps are performed in bulk [4,[17][18][19][20]. From an application point of view, devices of the first category offer an ideal method for direct quantification of digital assays but require the use of specialized equipment.…”

Section: Introductionmentioning

confidence: 99%

“…For off-chip solutions, many different droplet generation systems exist [4,[17][18][19][20]. However, most of them are difficult to manufacture, require complex experimental set-ups or need mineral oil for droplet generation [17][18][19].…”

Section: Introductionmentioning

confidence: 99%

“…However, most of them are difficult to manufacture, require complex experimental set-ups or need mineral oil for droplet generation [17][18][19]. The implementation of centrifugal microfluidics [25] enabled droplet generation on standard centrifuges without the need for any external pressure pumps, tubings, fittings or specialized devices, as it is the case for pressure driven systems [4,20]. But the so far developed systems [17][18][19] use mineral oil for droplet generation which has unfavorable properties compared to fluorinated oil, which is preferred for biochemical assays due to its high biocompatibility [26].…”

Section: Introductionmentioning

confidence: 99%

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Versatile Tool for Droplet Generation in Standard Reaction Tubes by Centrifugal Step Emulsification

et al. 2020

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We present a versatile tool for the generation of monodisperse water-in-fluorinated-oil droplets in standard reaction tubes by centrifugal step emulsification. The microfluidic cartridge is designed as an insert into a standard 2 mL reaction tube and can be processed in standard laboratory centrifuges. It allows for droplet generation and subsequent transfer for any downstream analysis or further use, does not need any specialized device, and manufacturing is simple because it consists of two parts only: A structured substrate and a sealing foil. The design of the structured substrate is compatible to injection molding to allow manufacturing at large scale. Droplets are generated in fluorinated oil and collected in the reaction tube for subsequent analysis. For sample sizes up to 100 µL with a viscosity range of 1 mPa·s-4 mPa·s, we demonstrate stable droplet generation and transfer of more than 6 × 10 5 monodisperse droplets (droplet diameter 66 µm ± 3 µm, CV ≤ 4%) in less than 10 min. With two application examples, a digital droplet polymerase chain reaction (ddPCR) and digital droplet loop mediated isothermal amplification (ddLAMP), we demonstrate the compatibility of the droplet production for two main amplification techniques. Both applications show a high degree of linearity (ddPCR: R 2 ≥ 0.994; ddLAMP: R 2 ≥ 0.998), which demonstrates that the cartridge and the droplet generation method do not compromise assay performance. resistance than the sample supply channel (ii), as long as the viscosity of the sample remains ≥ 4 mPa·s. Details of the fluidic design calculations and channel and chamber dimensions are listed in ESI 2, S1.Molecules 2020, 25, x 3 of 11

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“…We recently reported a technology termed Integrated Comprehensive Droplet Digital Detection (IC 3D), which is capable of analyzing milliliters of droplets within minutes to identify target pathogens ( Figure 1) [23][24][25][26]. Here, we report a phenotypic droplet assay for β-lactamase-mediated antibiotic resistance.…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of β-Lactamase-Producing Bacteria Using the Integrated Comprehensive Droplet Digital Detection (IC 3D) System

Cherukury

Labanieh

et al. 2020

Sensors

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Antibiotic-resistant bacteria have emerged as an imminent global threat. The lack of rapid and sensitive diagnostic techniques leaves health care providers with inadequate resources for guiding therapy and risks the lives of patients. The traditional plate culturing methods for identifying antibiotic-resistant bacteria is laborious and time-consuming. Bulk PCR (Polymerase Chain Reaction) and qPCR are limited by poor detection sensitivity, which is critical for the early-stage detection of bloodstream infections. In this study, we introduce a technique for detecting β-lactamase-producing bacteria at single-cell sensitivity based on a commercial β-lactamase sensor (Fluorocillin), droplet microfluidics, and a custom 3D particle counter. Bacteria-containing samples were encapsulated within picoliter-sized droplets at the single-cell level and cultured within water-in-oil droplets containing antibiotics and the Fluorocillin sensor. Then, fluorescent droplets were digitally quantified with the 3D particle counter, which is capable of analyzing milliliter-scale volumes of collected droplets within 10 min. The fluorescence signal from single-colony droplets was detectable in less than 5 h, and the 3D scanning was performed in less than 10 min, which was significantly faster than conventional culture-based methods. In this approach, the limit of detection achieved was about 10 bacterial cells per mL of sample, and the turnaround time from sample to result was less than 6 h. This study demonstrates a promising strategy for the detection of β-lactamase-producing bacteria using the recently developed IC 3D system.

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Rapid bacterial detection and antibiotic susceptibility testing in whole blood using one-step, high throughput blood digital PCR

Abstract: We report a rapid diagnostic platform that integrates novel one-step blood droplet PCR assay and a high throughput droplet counting system to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood.

Cited by 87 publications

References 45 publications

Microfluidic systems for rapid antibiotic susceptibility tests (ASTs) at the single-cell level

Microfluidic systems for rapid antibiotic susceptibility tests (ASTs) at the single-cell level

Versatile Tool for Droplet Generation in Standard Reaction Tubes by Centrifugal Step Emulsification

Rapid Detection of β-Lactamase-Producing Bacteria Using the Integrated Comprehensive Droplet Digital Detection (IC 3D) System

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