Rapid detection of bacterial pathogens in blood through engineered phages-beads and integrated Real-Time PCR into MicroChip
| Autor: | Sabrina Conoci, Salvatore P.P. Guglielmino, L.M. De Plano, S. Carnazza, Marco Sebastiano Nicolò, Salvatore Petralia, Concetta Gugliandolo, Vincenzo Zammuto, Domenico Franco, Maria Rizzo, Giovanna Calabrese |
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| Rok vydání: | 2021 |
| Předmět: |
02 engineering and technology
010402 general chemistry medicine.disease_cause 01 natural sciences Microbiology Sepsis Staphylococcus epidermidis Multiplex polymerase chain reaction Materials Chemistry medicine Electrical and Electronic Engineering Bacterial identification Instrumentation Escherichia coli biology Pseudomonas aeruginosa Chemistry Metals and Alloys Silicon microchip multiplex PCR 021001 nanoscience & nanotechnology Condensed Matter Physics medicine.disease biology.organism_classification 0104 chemical sciences Surfaces Coatings and Films Electronic Optical and Magnetic Materials Phage display Real-time PCR Real-time polymerase chain reaction Staphylococcus aureus 0210 nano-technology Bacteria |
| Zdroj: | Sensors and Actuators B: Chemical. 329:129227 |
| ISSN: | 0925-4005 |
| DOI: | 10.1016/j.snb.2020.129227 |
| Popis: | The invasion of pathogens into the bloodstream can produce a systemic inflammatory response syndrome that, if not promptly treated, rapidly evolves in acute life-threatening dysfunction of remote organs (septic shock). Current conventional diagnostic tests are microbial blood cultures that need days for results. Therefore, it is crucial the availability of rapid and reliable pathogens detection for prompt diagnosis. In this work, we developed a new method for fast and sensitive detection of bacteria in blood based on the integration of pathogen capture through engineered M13 phages and molecular detection via PCR in miniaturized silicon microchip. Engineered M13 phage-clones exposing specific peptides able to bind Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and a novel identified phage clone, 9IIIB5, for Staphylococcus epidermidis, were used to functionalize magnetic beads and capturing/concentrate the pathogens. Due to the stability of beads-phage-bacteria complex, PCR inhibitors present in the blood were easily removed without detachment of captured bacteria. The complex was used as target for direct amplification into miniaturized silicon chip by a multiplex PCR followed by Real-Time PCR. The procedure was very fast (less than 3 hs) and sensitive (10 cells/reaction) enabling molecular detection inside to the critical period for clinical decision in sepsis. It could be applied to detect pathogens present in very few numbers or in a metabolically inactive state. The results here presented pay the way to future development of portable genetic Point-of-Care in the rapid identification of microorganisms present in the blood stream or also in different matrices, such as waters and foods. |
| Databáze: | OpenAIRE |
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