Selection And Application Of Aptamers For Streptococcus

SELEX (Systematic Evolution of Ligands by Exponential Enrichment)， is a newcombinatorial chemistry technique which was invented successfully in the90s. Aptamers aresingle-stranded oligonucleotides that bind to target molecules with high affinity andspecificity，which is selected by repeated rounds from a synthetic random ssDNA pool bySELEX. At present，it is reported that various detection methods have been developed forfood-borne pathogens，but each has their own advantages and disadvantages. The studygained the aptamers of common pathogens (Streptococcus pyogenes and Streptococcusagalactiae) by Cell-SELEX respectively， and developed novel methods for pathogensdetection based on aptamer recognition.Firstly，a80-nt random single-stranded DNA pool containing40random sequences wassubjected to repeated twelve rounds of selection against Streptococcus pyogenes byCell-SELEX，negative SELEX and counter SELEX. Then，the selected oligonucleotides werecloned and sequenced. Analyzed the primary and secondary structures of the aptamersutilizing DNAMAN and RNA structure software. FAM-labeling aptamers were used foranalyzing binding affinity and specificity between the aptamers and Streptococcus pyogenesby Flow Cytometry Facility. There are two high affinity，specificity of aptamers selected forStreptococcus pyogenes. The dissociation constant is44.25±5.203nmol/L，53.89±7.586nmol/Lrespectively，and the average percent gated fluorescence intensity is77.05%，72.65%.Meanwhile，we also targeted Streptococcus agalactiae and synthetized a single-strandedDNA pool of the same length，different primers. Similar to selection and detection method ofStreptococcus pyogenes，we gained seven aptamers binding to Streptococcus agalactiae.Labeling them with FAM，we obtained two high affinity，high specificity aptamers by flowcytometry. The average percent gated fluorescence intensity is77.33%，80.95%respectively，and the dissociation constant is78.27±12.85nmol/L，49.88±6.871nmol/L.Then，a sensitive and rapid fluorescence method of detecting foodborne pathogens is setup，which is based upon magnetic beads-aptamers complex as capture probe，FAM-aptamersas fluorescence probe. The results showed that under the selected conditions，the colonyforming units of Streptococcus pyogenes were linear to the fluorescent intensity ranged from70to7100cfu/mL with a detection limit70cfu/mL. The standard recoveries were83.9%-103.3%for milk samples. The colony forming units of Streptococcus agalactiae werelinear to the fluorescent intensity ranged from50to5200cfu/mL with a detection limit50cfu/mL. The standard recoveries were91.1%-99.3%for milk samples.