Study On Novel Methods For Visual Detection Of Salmonella

Salmonella is one of the key target substances in food safety detection, thus, testingSalmonella in a fast and accurate way has important significance. Traditional detectionmethods of Salmonella would cost4~7days, cannot achieve the short-term test towards foodsafety. Currently, various detection methods have been developed for Salmonella, such asmultiplex PCR, Enzyme-linked immunosorbent assay(ELISA), and Loop-mediatedisothermal amplification(LAMP). However, most of these new methods have shortcomings insensitivity, specificity and facility of detection, some methods need to use expensiveequipments, which makes it different in promoting in basic level. Directed towards problemsabove, based on tyramine signal amplification and aptamers recognition coupled withimmunogold silver enhancement respectively, this study proposed novel methods for visualdetection of Salmonella.First, this study used Salmonella DNA as the target of detection, proposed a method forvisual detection of Salmonella based on oligonucleotide probe hybridization, tyramine signalamplification and color reaction of horseradish peroxidase（HRP) substrate. Oligonucleotideprobe was fixed in microwell plates by biotin-avidin system. Then, a large amount ofboitin-conjugated tyramine was deposited at the site of enzyme reaction under HRP catalysis.TMB was added and color reaction was performed. Under optimal conditions, the linear rangecovers a large variation of target concentration from1pmol/L to1000pmol/L with adetection limit of0.5pmol/L is obtained.Secondly, this study atampted to use the Salmonella bacteria as the target of detectiondirectly to develop another simple, sensitive, and rapid visual detection method based uponaptamer recognition coupled with immunogold silver enhancement. The complex ofavidin-biotinylated Salmonella Aptamer-Salmonella-gold namoparticles labelledhydrosulphonyl modified aptamer was assembled onto the microplate wells. After beinganchored on the complex through covalent Au-S bond, the signals of gold nanoparticles weresubsequently amplified by silver enhancement. Absorbance value was detected at630nm.Under optimal conditions, the correlation between the concentration of S.Typhimurium andthe absorbance value was found to be linear within the range of101~105cfu/mL (R2=0.9965),the limit of detection of the developed method was found to be10cfu/mL. The ability of thebioassay to detect S. Typhimurium in real water samples was also investigated, and the resultswere compared to the experimental results from the plate-counting methods. In addition, thisstudy explored the effect of silver enhancement solution and reaction conditions to the silverenhancement procedures, and prepared homemade silver enhancement solution which cangenerate similar detection results compared with commercially available reagents, thisresearch also discovered that silver enhancement strongly depends on the dispersed states ofgold nanoparticles.