The Rapid Detection Of Salmonella Based On Aptamer Signal Amplification

As a group of common gram-negative pathogenic bacteria,Salmonella threaten human health and bring tremendous economic loses to the livestock industry. It can not only cause typhoid fever, acute gastroenteritis, bacteremia and septicemia, but also cause widespread infection even death of livestock.Therefore, effective and efficient detection of Salmonella has been a hotspot in the clinic microbiology.Aptamer is a short DNA or RNA sequence derived from a random nucleotide library via systematic evolution of ligands by exponential enrichment(SELEX). It can specifically bind to a certain target. Since it has many advantages like broad spectrum of targets, high specificity and affinity, short period of screening cycle, aptamer has been applied to many fields. As a lately discovered nano carbon material, Graphene,has excellent mechanical, chemical and electrical properties and promising application potential in all fields.Hybridization chain reaction(HCR) can extend DNA strands without enzyme. The mild and simple reaction condition of HCR made it very suitable for the signal amplification of biosensors.The purpose of this work is to design sensitive, specificand efficient detection method of Salmonella enteritidis and S.typhimurium. The main works are displayed as follows:(1) A sensitive and selective aptasensor for the fluorimetric determination of S.enteritidis was developed by using Graphene Oxide and Klenow Fragment. In the absence of target, the aptasensor attached to GO and did not fluoresce due to the quenching effect of GO. When targets were introduced,the aptasensor bound to the target 16 S rRNA sequence and fluorescence signal was restored again. Meanwhile, both of the Klenow enzyme and the synergistic effects of FAM and SYBR Green I would help amplifying the fluorescence signal, which greatly improved the sensitivity of this method. The analyte of 16 SrRNA was determined over a linear range of 60 pM to 1nM with a detection limit of 60 pM. And it also exhibited a linear relationship with the concentration of S. enteritidis from 50 to 4×103CFU·mL-1, with a detection limit of 102CFU·mL-1in milk sample.(2) By means of the signal amplification by HCR, another aptasensor for swift, convenient and specific detection of S.typhimurium was constructed as well. The trigger sequence will expose when aptamer specifically binds to its target, and the exposed trigger sequence will initiates HCR. As it does not require enzyme and temperature alteration, the whole process condition of HCR was greatly simplified. All the results showedthat this aptasensor can achieve specific detection of Salmonella typhimurium in the linear range of 103-108CFU·mL-1with a detection limit of 103CFU·mL-1.