The Biosensor For Detection Of Salmonella Based On Rolling Circle Amplification

People have been plagued by the food safety in the world. Foodborne pathogens contamination for food led to hazard human health which restricting the development of the economy. Foodborne illnesses continuously threaten public health especially Salmonella typhimurium. This research describes hazards of the Salmonella, methods of detection for Salmonella. This research also describes the characteristics and functions of aptamer and rolling circles amplification. This research is composed of two systems ultrasensitive detection of Salmonella. The innovation of this research lies in the use of aptabiosensor and combines rolling circle amplification coupled DNAzyme to enhance the signal. Moreover, this research prepares silver nanoclusters, owing to its strong fluorescence signal.This research studies done as follows:Part one: In this plan, a simple, low-cost and very fast detection method is used to effectively select and detect the Salmonella typhimurium. This method is based on the nucleic acid fit body for the specificity and recognition and capture capabilities of Salmonella typhimurium. This experiment designed an arch probe, part of which is the base sequence of nucleic acids adapter, and the complementary strand is the primer amplification for rolling amplification. When the target appears in the system, nucleic acid fit body part of the arch probe captures the target by structural phase transformation, then primers were released. After adding the padlock probe and T4 DNA ligase in the system, padlock probe and primer combined together. When adding dNTP and phi29 DNA polymerase into the system, roll amplification process will take place, producing a long single-stranded DNA with innumerable the same DNA pieces, these units combined with hairpin structure whose both ends respectively modified fluorescent FAM and quenching reactive, then open the card. The hairpin sequence includes the cutting locus of the restriction enzymes, when issuing to roll in long chains of DNA amplification, restriction enzyme card will cut off the card, because of the combinations of base are too few, the cut hair cards fell down from the long chain. First, it will release the fluorescent, producing very strong fluorescent signals. Second, long chain can be recycled, reaching the effect of signal amplification. In the appropriate environment,the biosensor could ultrasensitively detect the Salmonella typhimurium with minimum lines of 7.8 cfu/mL, and the range of detection is four orders of magnitude. In addition, the biosensor shows very high selectivity for the targets, and has many advantages, such as fast detecting speed, low cost, etc. Which can be used as a useful tool for detection of Salmonella typhimurium in the actual samples and clinical diagnosis.Part two: In this work, a simple, low cost method for highly sensitive and selective detection of Salmonella typhimurium has been developed on the basis of exponential rolling circle amplification(ERCA) coupled DNAzyme amplification. A hairpin containing Salmonella typhimurium aptamer and a primer sequence complementary to a padlock probe, which includes one Ag clusters units, is used for recognizing Salmonella typhimurium and triggeringERCA. Padlock probe were composed of Ag clusters units and nicking site. Due to ERCA coupled DNAzyme amplification strategy, the presence of Salmonella typhimurium leads to open hairpin and expose to the primer, which complementary to padlock probe. Add DNA polymerase and so on to the system to generate a lot of unite to incorporate Ag clusters. It generates a quantity of Ag clusters and gives a remarkably strong fluorescence response. Under optimal conditions, the proposed biosensor exhibits ultrasensitive toward Salmonella typhimurium with detection limits of 10 cfu/m L and a detection range of 4 orders of magnitude. Besides, our method also shows high selectivity toward target Salmonella typhimurium and has the advantages in its rapidness, low cost, simplified operations Hence, the RCA coupled nicking DNAzyme amplification-based fluorescence method might become a practical and useful for detecting Salmonella typhimurium.