A nanoparticle-based method for culture-free bacterial DNA enrichment from whole blood

Hassan, Marwa M.; Ranzoni, Andrea; Cooper, Matthew A.

doi:10.1016/j.bios.2017.07.057

Cited by 26 publications

(21 citation statements)

References 49 publications

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“…IgG in the blood also interferes with the activation of Taq polymerase as it interacts with single-strand DNA at high temperatures [ 24 ]. To minimize this inhibitory effect on molecular amplification, users have to use commercial kits and systems for purifying bacterial DNA from blood [ 25 , 26 , 27 , 28 ]. The QIAamp blood mini kit can purify DNA in 200 µL of blood without any dilution but cannot differentiate bacterial DNA from the DNA of blood cells.…”

Section: Resultsmentioning

confidence: 99%

A 3D-Printed Millifluidic Platform Enabling Bacterial Preconcentration and DNA Purification for Molecular Detection of Pathogens in Blood

2018

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Molecular detection of pathogens in clinical samples often requires pretreatment techniques, including immunomagnetic separation and magnetic silica-bead-based DNA purification to obtain the purified DNA of pathogens. These two techniques usually rely on handling small tubes containing a few millilitres of the sample and manual operation, implying that an automated system encompassing both techniques is needed for larger quantities of the samples. Here, we report a three-dimensional (3D)-printed millifluidic platform that enables bacterial preconcentration and genomic DNA (gDNA) purification for improving the molecular detection of target pathogens in blood samples. The device consists of two millichannels and one chamber, which can be used to preconcentrate pathogens bound to antibody-conjugated magnetic nanoparticles (Ab-MNPs) and subsequently extract gDNA using magnetic silica beads (MSBs) in a sequential manner. The platform was able to preconcentrate very low concentrations (1–1000 colony forming units (CFU)) of Escherichia coli O157:H7 and extract their genomic DNA in 10 mL of buffer and 10% blood within 30 min. The performance of the platform was verified by detecting as low as 1 CFU of E. coli O157:H7 in 10% blood using either polymerase chain reaction (PCR) with post gel electrophoresis or quantitative PCR. The results suggest that the 3D-printed millifluidic platform is highly useful for lowering the limitations on molecular detection in blood by preconcentrating the target pathogen and isolating its DNA in a large volume of the sample.

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Section: Resultsmentioning

confidence: 99%

A 3D-Printed Millifluidic Platform Enabling Bacterial Preconcentration and DNA Purification for Molecular Detection of Pathogens in Blood

2018

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“…Very few phage-based assays have been described in the literature for the detection of bacteria directly from blood [72][73][74] . Our implemented method presents several advantages over others which require extensive sample pre-treatment [74][75][76] , genetic manipulation of phage genomes 73 , complex instrumentation 22,[75][76][77] , expensive biorecognition molecules 22,43,77,78 or entail nucleic acids extraction to complete the detection [74][75][76] .…”

Section: Discussionmentioning

confidence: 99%

A novel flow cytometry assay based on bacteriophage-derived proteins for Staphylococcus detection in blood

Costa

Dias

Melo

et al. 2020

Sci Rep

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Bloodstream infections (BSIs) are considered a major cause of death worldwide. Staphylococcus spp. are one of the most BSIs prevalent bacteria, classified as high priority due to the increasing multidrug resistant strains. Thus, a fast, specific and sensitive method for detection of these pathogens is of extreme importance. In this study, we have designed a novel assay for detection of Staphylococcus in blood culture samples, which combines the advantages of a phage endolysin cell wall binding domain (CBD) as a specific probe with the accuracy and high-throughput of flow cytometry techniques. In order to select the biorecognition molecule, three different truncations of the C-terminus of Staphylococcus phage endolysin E-LM12, namely the amidase (AMI), SH3 and amidase+SH3 (AMI_SH3) were cloned fused with a green fluorescent protein. From these, a higher binding efficiency to Staphylococcus cells was observed for AMI_SH3, indicating that the amidase domain possibly contributes to a more efficient binding of the SH3 domain. The novel phage endolysin-based flow cytometry assay provided highly reliable and specific detection of 1-5 CFU of Staphylococcus in 10 mL of spiked blood, after 16 hours of enrichment culture. Overall, the method developed herein presents advantages over the standard BSIs diagnostic methods, potentially contributing to an early and effective treatment of BSIs. Bloodstream infections (BSIs) are severe diseases caused by the presence of microorganisms, mainly bacteria, in blood and are characterized by high morbidity and mortality 1,2. The BSIs and associated organ dysfunctions (sepsis or septic shock) remain a life-threating disease due, partially, to the inability to rapidly detect and identify the responsible pathogens 3,4. Staphylococcus spp. are Gram-positive facultative anaerobic bacteria that frequently colonize the human body 5,6. These pathogens are becoming increasingly resistant to antibiotics and are well-established in both community and healthcare environments, being commonly isolated in intensive care units (ICU) 6,7. Staphylococcus aureus is a common cause of a variety of infections, from superficial skin infections to life-threatening diseases, including necrotizing pneumonia 8 , infective endocarditis 9 and BSIs 10. Coagulase-negative staphylococci (CoNS) have also been described as harmful to humans, causing several infections, particularly in patients with implanted medical devices 6. The empirical antibiotic therapy remains the standard of BSIs treatments 11 and its correct use within the first hour after the recognition of the BSI is recommended by the Surviving Sepsis Campaign Guidelines 11 and was reported as having a great impact on the patient survival rate 12. Nevertheless, the extensive use of broad-spectrum antibiotics and the large number of patients having negative blood culture samples and thus receiving unnecessary antibiotic treatment, are important contributors to the increase of antimicrobial resistance 13-15. Thus, sensitive, rapid, cost-efficient and specific ...

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“…Li [ 58 ] realized the inertial focusing and ordering of Euglena gracilis using a microfluidic device consisting of a straight rectangular microchannel, a gradually expanding region, and five outlets with fluidic resistors to enhance an accurate separation ( Figure 6 c). Hassan [ 59 ] reported on the development of a culture-free bacterial enrichment method to concentrate bacteria from whole blood. This can concentrate bacterial cells and their DNA with a 500-fold reduction in sample volume.…”

Section: Progress In Inertial Microfluidicsmentioning

confidence: 99%

Progress of Inertial Microfluidics in Principle and Application

Gou

Jia

Wang

et al. 2018

Sensors

137

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Inertial microfluidics has become a popular topic in microfluidics research for its good performance in particle manipulation and its advantages of simple structure, high throughput, and freedom from an external field. Compared with traditional microfluidic devices, the flow field in inertial microfluidics is between Stokes state and turbulence, whereas the flow is still regarded as laminar. However, many mechanical effects induced by the inertial effect are difficult to observe in traditional microfluidics, making particle motion analysis in inertial microfluidics more complicated. In recent years, the inertial migration effect in straight and curved channels has been explored theoretically and experimentally to realize on-chip manipulation with extensive applications from the ordinary manipulation of particles to biochemical analysis. In this review, the latest theoretical achievements and force analyses of inertial microfluidics and its development process are introduced, and its applications in circulating tumor cells, exosomes, DNA, and other biological particles are summarized. Finally, the future development of inertial microfluidics is discussed. Owing to its special advantages in particle manipulation, inertial microfluidics will play a more important role in integrated biochips and biomolecule analysis.

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A nanoparticle-based method for culture-free bacterial DNA enrichment from whole blood

Cited by 26 publications

References 49 publications

A 3D-Printed Millifluidic Platform Enabling Bacterial Preconcentration and DNA Purification for Molecular Detection of Pathogens in Blood

A 3D-Printed Millifluidic Platform Enabling Bacterial Preconcentration and DNA Purification for Molecular Detection of Pathogens in Blood

A novel flow cytometry assay based on bacteriophage-derived proteins for Staphylococcus detection in blood

Progress of Inertial Microfluidics in Principle and Application

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