Research On Aptamer Selection And Application Of Enterohemorrhagic Escherichia Coli O157:H7

Escherichia coli O157:H7 is a bacterial pathogen that causes gastrointestinal bleeding of human and livestock,and is a typical food-borne pathogen.Cattle are one of the main carriers of this bacterial pathogen in nature,which usually carry asymptomatically.Human and poultry are also the carriers of this bacterial pathogen.The main route of transmission of this bacteria is generally believed to be fecal-oral route,and there are a wide range of media,including contaminated water sources,soil,meat products,eggs,as well as milk and so on.Escherichia coli O157:H7 is highly infectious with a low infectious dose.There have been reports of epidemics and sporadic cases of it in most parts of the world,which aroused a widespread concern.The traditional methods for detecting the presence of bacterial have the unavoidable shortcomings of long detection time,low detection sensitivity and cumbersome experimental operation steps,which are not suitable for rapid detection in the field.Aptamer is a single-stranded DNA or RNA that has high affinity and high specificity for its target substance,which is screened by SELEX(Systematic Evolution of Ligands by Exponential enrichment)technology in vitro.It has a wide range of target substances,short screening time,high affinity,high specificity,easy to save or transport,and many other advantages,which is regarded as a substitute for antibodies.Surface enhanced Raman spectroscopy(SERS),refers to the phenomenon of greatly enhancing the Raman signal intensity by using a special surface-enhanced adsorption molecule,which has been widely applied in the field of bacterial detection at present.The research contents are as follows:(1)Screening and evaluating specific aptamer of Escherichia coli O157:H7.As a target substance,Escherichia coli O157:H7 was incubated with a random oligonucleotide bank to isolate the unbound ssDNA,and enrich bound ssDNA.After fifteen rounds of screening process,a high affinity and high specificity aptamer library could be obtained.Candidate aptamer sequences were obtained by high throughput sequencing and secondary structure analysis,and were analyzed for affinity and specificity by ELISA techniques.Then the aptamers with high affinity and high specificity to the target substance were obtained.(2)The establishment of a rapid and efficient method for the detection of Escherichia coli O157:H7 based on aptamer-mediated SERS.Escherichia coli O157:H7 was incubated with its specific folded aptamers,the unbound and the less bound aptamers were separated by washing.Using the method of reducing nano silver in situ,the silver was reduced on the surface of the complex combined by Escherichia coli O157:H7 and its specific aptamers.Raman signals were acquired by Raman spectrometer.Whole-bacteria-SELEX technology was applied in this study,and the ssDNA aptamers of Escherichia coli O157:H7 with high affinity and high specificity were obtained,which lay the foundations for the detection methods of Escherichia coli O157:H7.This study was based on aptamer-mediated SERS technique,established a rapid and simple detection method of Escherichia coli O157:H7 with high specificity and sensitivity.This method has good repeatability and the lowest detection limit is 10~2cfu/mL,which has a certain developable prospect.