The Selection And Application Of Aptamers Against Different Stages Of E.Coli O157:H7

Escherichia coli O157:H7(E.coli O157:H7)is an important foodborne pathogen that causes serious public health hazards worldwide.It is the most studied serotype among Enterohemorrhagic Escherichia Coli.The traditional E.coli O157:H7 detection method has defects in detection time,sensitivity and specificity,which makes it can not meet increasingly strict detection requirements.Therefore,it is very important to develope a rapid,sensitive and economical E.coli O157:H7 detection and analysis method.Aptamer is an ssDNA or RNA that can combine with wide range of targets specificity and affinity.As a new kind of nanoporous material,Cu-MOF has many special properties such as porosity and specific surface area,biocompatibility,biomimetic catalytic properties,etc.It has been applied in many fields such as medicine,chemical industry,food,etc.In this study,we combined Cu-MOF with aptamers for the detection of E.coli O157:H7 using its catalytic properties,based on the aptamers of E.coli O157:H7.The main work is as follows:Firstly,E.coli O157:H7 aptamers were screened based on the Cell-SELEX technique.In this study,E.coli O157:H7(adjustment phase,logarithmic phase and stationary phase)at different growth stages were used as positive targets while Shigella flexneri,Enterotoxigenic Escherichia Coli,Salmonella typhimurium and Escherichia coli,Staphylococcus aureus as negtive strain.Through incubating with ssDNA librabry,separating the ssDNA that cannot bind with E.coli O157:H7,dissociating the ssDNA bind with E.coli O157:H7 and PCR,enzyme digestion and purification steps,after 11 positive screening and 3 negtive screening,the ssDNA library with better binding of E.coli O157:H7 was collected gradually,and the14th round of PCR product was cloned and sequenced.Secondly,the aptamer affinity and specificity verification and its binding mechanism were studied.Thirty-two oligonucleotide sequences obtained from the sequencing of the clones were analyzed.The 32 sequences were divided into nine families according to their homology and secondary structure.A representative sequence from each family was selected for affinity and specificity analysis.By flow cytometry analysis,we obtained the best two aptamers against E.coli O157:H7 with the binding fluorescence intensity was 76.94%and77.29%,respectively.The K_d value was 9.04±2.08 nmol/L and 15.13±0.88 nmol/L,respectively.Then treat E.coli O157:H7 with proteinase K and trypsin.The fluorescence intensity was measured by incubation of E.coli O157:H7 with carboxyfluorescein(FAM)-labeled aptamers.The binding fluorescence intensity did not decrease but increased slightly,it was proved that the binding site was not bacterial membrane protein.Finally,the method that Cu-MOF as a peroxidase mimetics for colorimetric detection of E.coli O157:H7 was conducted.One biotin-labeled aptamer was fixed on the microplate as capture probe and another one linked with Cu-MOF as displayed probe.The results showed that E.coli O157:H7 had a good linear relationship with absorbance at 450 nm in the range of42~4.2×10~6 cfu/m L.The linear regression equation was y=0.12089x+0.01176(R~2=0.99414).The limit of detection is 31 cfu/mL.The actual milk samples were detected by this method.There was no significant difference between the results and the plate count method test results,indicating that the method is practical and reliable.