Designing a non-enzymatic system at constant roomtemperature as a signal amplification technique for the detection ofKlebsiella pneumoniae

Background and Aim : Encapsulated, gram negative, and non-motile bacilli Klebsiellapneumoniae is one of the most common secondary infection agents enlisted as a serious threat bythe CDC according to its wide-range drug resistance. Amid vast molecular diagnostic tools, nanobiosensingis a proper candidate to provide simplicity and accuracy simultaneously. Herein, asignal amplification autonomous enzyme-free machinery was designed to sense a virulent gene inKlebsiella pneumoniae specifically.Methods : A target gene in Klebsiella pneumoniae, responsible for the capsule diversity, wasselected. Two oligonucleotides as the main components of the system were created and verifiedby particular online tools. The gene was added to the reaction mixture as a trigger. The signalamplification process was conducted at a constant room temperature. During a one-hour incubationat ۲۵ ℃, color differentiation between the positive and the negative samples was monitored. Thedistance between nanostructures was measured by TEM imaging.Results : The results demonstrated that the nanostructures were more aggregated when the targetgene was absent. However, a step-by-step reaction cascade was conducted in the presence of thetarget, leading to more relative stability in the solution, which was caused by the further distancebetween the secondary structures (that are only formed when the amplification is implemented).The visually detectable more-reddish color of the positive samples was interpreted as a result ofthe abovementioned stability, which was confirmed by TEM images.Conclusion : The selected gene, which is only present in K. pneumoniae, plays a role in itspathogenicity and antigenicity, meaning that the gene is worth diagnosing and it guarantees thespecificity of the method. The developed technique could specifically sense the pathogenic nucleobiomarkerand amplify the signal at room temperature, needless of any enzyme and sophisticatedinstrumentation. The fact was confirmed by both in-silico and in-vitro analyses.

کلیدواژه ها:

Signal amplification ، Nucleo-biomarker ، Direct diagnosis ، Klebsiella pneumoniae