Rapid Detection Of Shigella Flexneri Using Signal Amplification Technique Based On Aptamer

Shigella is a genus of bacteria that are a major cause of diarrhea and dysentery throughout the world, which causes roughly 120 millions of dysentery infections and kills over 1.1 millions of people (predominantly young children) worldwide every year. Antibiotics are the standard of care for shigellosis, but therapeutic options are limited by the widespread prevalence of resistant strains, as in Asia where resistance to ciprofloxacin has become common. Considering the difficulties in treating and preventing shigellosis, the development of simple, sensitive and selective methods for monitoring the pathogen is therefore in ever-increasing demand. Since Shigella flexneri are the major agents of endemic shigellosis among children in developing countries, they were chosen as target bacteria in this experiment.Graphene oxide (GO), a two-dimensional carbon crystal with only one carbon atom thickness, has attracted extensively attention due to its exceptional biocompatibility, high surface-to-volume ratio, high electron transfer rate. Specifically, owing to its extraordinary and distance-dependent fluorescence quenching property, it was proposed as a universal highly efficient long-range quencher. In addition, it can interact with single-stranded DNA noncovalently through π-π stacking interactions, while hardly interact with rigid double-stranded DNA or aptamer-target complexes. It can also protect single-stranded DNA against enzymatic cleavage. Hence, GO exhibit particularly enormous potential application in disease diagnosis and biological analysis.Aptamers are single-stranded artificial oligonucleotides (DNA or RNA) separated from random-sequence nucleic acid libraries through an in vitro selection process termed as the systematic evolution of ligands by exponential enrichment (SELEX). They exhibit high affinity and specificity towards their targets including protein, organic molecules, various cell surface receptors, and whole cells. Due to their superior advantages in terms of temperature stability, low cost, reusability, easy synthesis and modification over antibodies, aptamers have been increasingly used as recognition probes for designing different types of biosensors. Meanwhile, the aptamer-target recognition event can be easily amplified by nuclease based signal amplification strategy, making an extraordinarily low detection limit possible. Hence, aptamers are an ideal method for the detection of food-borne pathogens, especially bacteria, in environment, food or clinical samples.In this thesis, by selecting S. flexneri as the target, we carried out the research work focused on highly sensitivity and selectivity pathogen detection. The main works are summarized as follows:1. Aptamer selection and identificationA panel of single-strand DNA aptamers with high affinity and specificity against the S. flexneri were selected from a combinatorial DNA library, then they were sorted, cloned and sequenced. Three single-strand DNA were chosen and nominated aptamer 1,2,3. Compared with the other two candidate aptamers, aptamerl was chosen for further experiments as it had highly binding affinity with an apparent dissociation constant (Kd value) of 29±4 nM.2. Aptasensor for detecting bacteria by using signal amplification technique based on aptamerBased on enzyme-assisted signal amplification technique, a simple, sensitive, selective and homogeneous fluorescent aptasensor was constructed for S. flexneri by using dye-labeled aptamer and GO. In this aptasensor, the unique recognition sequences of restriction endonuclease ApaI were incorporated into the single-stranded signal probe which labeled with carboxyfluorescein at its 5’and 3’end. In the absence of target bacteria, probes were adsorbed and quenched effectively by GO. After the addition of the target, the specifically binding of aptamer to target liberated the probes from GO, and the associated probes were recognized and cleaved by this enzyme. In addition, the released targets were used again in subsequent rounds. Under the optimum conditions, a linear relationship was displayed in 500 to 108 CFU/mL with a low limit of 100 CFU/mL and high selectivity. Consequently, this aptasensor should be a potential alternative to the conventional detection ones.