Direct droplet digital PCR (dddPCR) for species specific, accurate and precise quantification of bacteria in mixed samples

Pacocha, Natalia; Scheler, Ott; Nowak, Mikolaj Marcin; Derzsi, Ladislav; Cichy, Joanna; Garstecki, Piotr

doi:10.1039/c9ay01874c

Cited by 14 publications

(11 citation statements)

References 29 publications

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“…2 ). For subsequent sequencing, bacterial cell lysis by heating 35 , amplifications of barcode and 16S rRNA genes (V3–V4 region, ~450 bases), attachment of sequencing adapters, and linkage 36 between amplified barcodes and 16S rRNA genes were performed in a single step (Fig. 1c and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

High-throughput identification and quantification of single bacterial cells in the microbiota

et al. 2022

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The bacterial microbiota works as a community that consists of many individual organisms, i.e., cells. To fully understand the function of bacterial microbiota, individual cells must be identified; however, it is difficult with current techniques. Here, we develop a method, Barcoding Bacteria for Identification and Quantification (BarBIQ), which classifies single bacterial cells into taxa–named herein cell-based operational taxonomy units (cOTUs)–based on cellularly barcoded 16S rRNA sequences with single-base accuracy, and quantifies the cell number for each cOTU in the microbiota in a high-throughput manner. We apply BarBIQ to murine cecal microbiotas and quantify in total 3.4 × 105 bacterial cells containing 810 cOTUs. Interestingly, we find location-dependent global differences in the cecal microbiota depending on the dietary vitamin A deficiency, and more differentially abundant cOTUs at the proximal location than the distal location. Importantly, these location differences are not clearly shown by conventional 16S rRNA gene-amplicon sequencing methods, which quantify the 16S rRNA genes, not the cells. Thus, BarBIQ enables microbiota characterization with the identification and quantification of individual constituent bacteria, which is a cornerstone for microbiota studies.

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Section: Resultsmentioning

confidence: 99%

High-throughput identification and quantification of single bacterial cells in the microbiota

et al. 2022

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“…Identification of pathogens in wastewater is of paramount importance. As described above, ddPCR is able to detect and quantitate pathogen DNA/RNA residues using the power of statistics, and therefore this method has been expectedly used in a similar way to measure the abundance of a specific microbe both in monocultures and in consortia [ 49 ] and of antimicrobial-resistant bacterial cells in blood samples [ 36 ] ( Fig. 2 B).…”

Section: Analysis and Monitoring Of Microorganisms In Dropletsmentioning

confidence: 99%

Droplet microfluidics on analysis of pathogenic microbes for wastewater-based epidemiology

Cao

Zhang

et al. 2021

TrAC Trends in Analytical Chemistry

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Infectious diseases caused by pathogenic microbes have posed a major health issue for the public, such as the ongoing COVID-19 global pandemic. In recent years, wastewater-based epidemiology (WBE) is emerging as an effective and unbiased method for monitoring public health. Despite its increasing importance, the advancement of WBE requires more competent and streamlined analytical platforms. Herein we discuss the interactions between WBE and droplet microfluidics, focusing on the analysis of pathogens in droplets, which is hard to be tackled by traditional analytical tools. We highlight research works from three aspects, namely, quantitation of pathogen biomarkers in droplets, single-cell analysis in droplets, and living cell biosensors in droplets, as well as providing future perspectives on the synergy between WBE and droplet microfluidics.

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“…Compounding the issue is the high incidence of drug-resistant bacteria and especially multi-drug resistant (MDR) bacteria, the prevalence of which is further increased by nonspecific broad-spectrum antibiotic administration [7,8]. Therefore, there is an urgent need for a rapid diagnostic test that can give sample-to-result in less than 24 h. More recently, nano-and micro-based systems have been developed such as Droplet Digital PCR (ddPCR) [9][10][11], which involves the partitioning of the sample into thousands or millions of droplets, followed by separate PCR reactions occurring within each individual droplet. The number of DNA copies contained in each droplet can be predicted by the Poisson distribution [12].…”

Section: Introductionmentioning

confidence: 99%

“…More recently, nano- and micro-based systems have been developed such as Droplet Digital PCR (ddPCR) [ 9 , 10 , 11 ], which involves the partitioning of the sample into thousands or millions of droplets, followed by separate PCR reactions occurring within each individual droplet. The number of DNA copies contained in each droplet can be predicted by the Poisson distribution [ 12 ].…”

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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Direct droplet digital PCR (dddPCR) for species specific, accurate and precise quantification of bacteria in mixed samples

Abstract: Here we demonstrate a direct droplet digital PCR assay (dddPCR) for simultaneous identification and quantification of bacteria in a bacterial mixture.

Cited by 14 publications

References 29 publications

High-throughput identification and quantification of single bacterial cells in the microbiota

High-throughput identification and quantification of single bacterial cells in the microbiota

Droplet microfluidics on analysis of pathogenic microbes for wastewater-based epidemiology

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

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