Selection And Applied Research Of Aptamers Targeted To Staphylococcus Aureus Subsp.aureus And Bacillius Cereus

SELEX is a novel technique of combinatorial chemistry developed in the early 1990 s. Aptamer is a short single-stranded oligonucleotide(DNA or RNA) that can bind to tar get material with high affinity and specificity in specific secondary structure, which is selected from an in vitro chemical synthesis random ss DN A or ss RNA pool by SELEX. The study obtained the aptamers of two kinds of food-borne pathogens(S. subsp.aureus and B. cereus) by SELEX, and established novel methods respectively for the detection of the pathogens based on aptamer recognition.Firstly, an 80 nt random ss DNA pool with a capacity of 1014~1015 was subjected respectively to fourteen rounds of selection against S. subsp.aureus and B. cereus by SELEX and counter SELEX. Then, the softwares of DNAMAN and RNAstructure were used to analyzed the primary and predicted secondary structures of the selected ss DNA. FAM- labeled aptamers were used for analyzing binding affinity and specificity between the aptamers and S. subsp.aureus or B. cereus by Flow C ytometry Facility. Finally, the study gained one aptamer specific to S. subsp.aureus. The dissociation constant is 26.79±9.88 nmol/L, and the gated fluorescence intensity is 79.1%; Meanwhile, the study gained two aptamers specific to B. cereus. Their dissociation constant is 14.09±4.83 nmol/L and 21.96±6.85 nmol/L, and their gated fluorescence intensity is 94.36% and 91.29%.Then, a rapid and sensitive fluorescent detection method of S. subsp.aureus was established using its biotin- labeled aptamer as capture probe and FAM- labeled aptamer as displayed probe. The results showed that under the optimum conditions, the colony forming units of S. subsp.aureus were linear to the fluorescence intensity ranged from 60 to 8300 cfu/m L with a detection limit of 60 cfu/m L. The standard recoveries from milk sample were 82.1%~108.9%.Finally, a label- free electrochemical impedamic sensor detection method for B. cereus was established using its SH-aptamer as capture probe and Au NPs as signal amplifier. The results showed that under the optimum conditions, the colony forming units of B. cereus were linear to the changes of the resistance ranged from 101 to 107 cfu/m L with a detection limit of 10 cfu/m L. The standard recoveries of milk sample were 93.8%~107.4%.