Screening And Application Of Enterococcus Faecalis-specific Single-stranded DNA Aptamers

Enterococcus is the second most common pathogen of endogenous and exogenous nosocomial infections,which can cause various diseases,such as meningitis,urethritis,sepsis and abdominal infection.With the frequent use of antibiotics,the multi-drug resistance of Enterococcus is increasing.Especially,the emergence of vancomycin-resistant Enterococcus makes Enterococcus infection become a public health problem that needs to be solved urgently.CHINET monitoring data show that Enterococcus faecalis?E.faecalis?is the main cause of infection.Thus it is of great significance to develop molecular probes with high affinity for E.faecalis which can be used in the identification and detection of E.faecalis.In this thesis,single-stranded DNA aptamers against E.faecalis were screened by whole-bacteria systematic evolution of ligands by exponential enrichment?whole-bacteria SELEX?after multiple rounds of screening.Then the structure and property of the selected aptamers were characterized.Combined with graphene oxide,the aptamer was used to establish a novel method for the detection of E.faecalis.Firstly,the whole-bacteria SELEX was carried out to screen aptamers with high affinity and high specificity for E.faecalis from a single-stranded DNA library with the length of 79nucleotides containing 35 random bases.During the SELEX process,the affinity of ssDNA sub-library toward E.faecalis was evaluated by fluorescence analysis.After 12 rounds of selection,the obtained ssDNA pool was cloned and sequenced,from which overall 39aptamers were screened.The nucleotide sequences of these aptamers were subjected to primary structure analysis and secondary structure simulation using DNAMAN software and Mfold software.According to the analysis results,they were divided into 7 families,from which 4 structurally stable,different family-source aptamers were selected as candidates.The values of dissociation constant?K_d?of Apt 10,Apt 21,Apt 29 and Apt 34 were determined by fluorescent analysis,which were 862.0±106.4 nmol·L^-1,549.2±147.4 nmol·L^-1,614.3±121.9nmol·L^-1 and 988.0±208.7 nmol·L^-1,respectively.Among them,Apt 21 shows the highest affinity for E.faecalis.The specificity of Apt 21 was also verified.The aptamer shows low binding ability to other pathogenic bacteria,indicating satisfactory specificity.Therefore,Apt21 was chosen as the optimal aptamer for E.faecalis.Secondly,Apt 21 was used as the recognition element to construct a fluorescent assay for detecting E.faecalis.Graphene oxide?GO?can adsorb FAM-labeled aptamer and quench the fluorescent signal.However,the target E.faecalis can competitively bind with the aptamer adsorbed on the surface of GO and recover the fluorescent signal,which can be utilize to detect the target.The detection conditions,including the amount of GO and the fluorescence quenching time were optimized,which were fixed as 20?L and 6 min.Under the optimal conditions,the detection of E.faecalis was carried out.In the range of 10³¹0⁷ cfu·mL^-1,the recovered fluorescence intensity increases with the increased concentration of E.faecalis,and a good linear relationship between the fluorescent intensity of the logarithm of the concentration of E.faecalis was derived.The detection limit of 41 cfu·mL^-1 was derived.Further verification of the specificity of the assay was also performed.Finally,the constructed fluorescent assay was successfully applied to detect E.faecalis in water samples.Therefore,the screened aptamers and the established assay for E.faecalis in this thesis have great application prospects in clinical diagnosis and disease treatment.