The Detection And Biofilm Control Of Salmonella Based On Aptamer

Salmonella is a common zoonotic pathogen through poultry and livestock spreading to human, giving rise to many infections, such as gastroenteritis, septicemia, typhoid fever and so on. And its main species are Salmonella paratyphi A, Salmonella choleraesuis, Salmonella enteritidis, Salmonella typhimurium. Biofilms formed by Salmonella exist widely in the host and natural environment, leading to difficult treating and recrudescing. Outbreaks of S. paratyphi A infection continue to occur worldwide and have drawn close attention, so a useful practical detection platform is essential to the early rapid diagnosis of the infection. S. choleraesuis infringes on weaned piglets under 4 months, causing intestines problems and direct economic loss for breeding industry. In addition, the formed biofilms aggravate further its tolerance. Therefore, it is significant to establish a new method for detecting Salmonella and contolling its biofilm.(1) Highly sensitive cyclic signal amplification detection for S. paratyphi A. In this study, a simple and cost-effective DNA aptasensor, which was composed of a designed aptamer(DA) and two short FAM-modified sequences(probe 1 and probe 2) for fluorimetric determination of the pathogen, was constructed via fluorescence superposition, fluorescence resonance energy transfer(FRET) and enzymolysis of restriction endonuclease. In the absence of target, the FAM-modified aptasensor was bound to graphene oxide(GO) and the FAM fluorescence was quenched. In the presence of target, however, the FAM-modified aptasensor was released from the surface of GO due to specific binding of the aptasensor to the target and a strong fluorescent signal could be detected. More importantly, the fluorescent signal could be substantially amplified by a DNase I-mediated target recycling process. Under the optimized conditions, a good linear relation was displayed from 1×102 to 1×1011 cells/mL, suggesting that the lowest detection limit was 1×102 cells/mL. This constructed detection platform exhibits a high sensitivity and specificity in the detection of the pathogen, and it may be a potential approach for detection of other bacteria.(2) The control of Aptamer-Ampicillin conjugate(Apt-Amp conjugate) on S. choleraesuis biofilm. Here, a synergetic protocol to control the biofilm of this pathogen was provided based on the amide interaction between molecular structures of aptamer and ampicillin. The bifunctional conjugate was synthesised by linking an amidogen-modified aptamer(aptamer 3) at the 3’- terminal specific to its flagellin to ampicillin through amidation reaction. This synthesis was confirmed by spectroscopy method and its specificity was also investigated by fluorescence spectrophotometry. Furthermore, the conjugate had great influence on the biofilm formation and control by virtue of ultraviolet-visible(UV) spectrophotometer, inverted microscopy and atomic force microscopy(AFM). The results demonstrated that the Apt-Amp conjugate had great effects on controlling biofim, which was able to inhibit the biofilm formation by 71.64% and eliminate the preformed biofilm by 60.00%. Notably, the effects were better than its compositions(aptamer, ampicillin). Therefore, it is proposed that the conjugate can function simultaneously as specific recognition of aptamer 3 and bacteriostasis of ampicillin and promote its inhibition and elimination for bacterial biofilms.