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.
Rapid detection of bacterial pathogens in blood through engineered phages-beads and integrated Real-Time PCR into MicroChip
Rizzo M. G.;Carnazza S.;De Plano L. M.;Franco D.;Nicolo M. S.;Zammuto V.;Calabrese G.;Gugliandolo C.;Conoci S.
;Guglielmino S. P. P.
2021-01-01
Abstract
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.Pubblicazioni consigliate
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