Selection And Tailoring Of Aptamers Of Cronobacter Sakazakii And Construction Of Functional Nucleic Acid Biosensor

Cronobacter sakazakii(C.sakazakii)belongs to the genus cronobacter.It is the conditional pathogen of necrotizing enterocolitis,septicemia and meningitis of all ages.Cronobacter sakazakii has been reported as the main cause of severe neonatal and infant infections in the genus cronobacter,and the outbreaks of these bacterial infections are mainly related to infant formula.Aptamer is a functional DNA/RNA structure with high affinity and specificity for small inorganic and organic molecules,proteins,whole cells and other targets,and is widely used in food detection,environmental detection,molecular diagnosis and other fields.However,a large number of aptamers screened out will inevitably have differences in affinity and specificity.Therefore,it is necessary to establish a reasonable and efficient aptamer performance evaluation system to select the most suitable aptamer,and then cut the selected aptamer to remove the redundant sequence and further improve the affinity of the aptamer.On this basis,the aptamer biosensor will be more sensitive,more efficient and more conducive to follow-up applications.For this reason,we established a performance evaluation system for aptamers of Cronobacter sakazakii by fluorescence qPCR,and tailor out the selected aptamers.It lays a solid foundation for the follow-up practical application.1.A comprehensive evaluation system was established to evaluate the aptamer performance of Cronobacter sakazakii in terms of affinity,specificity and ability to bind live/dead bacteria,and the most suitable original aptamer sequence was selected for the following aptamer tailoring work.2.The secondary structure of aptamer was predicted by "MFold" software,and cut according to the predicted secondary structure.The core sequence is obtained by truncation and base substitution,and the core sequence is optimized.3.The affinities of original aptamers,optimized aptamers and reported aptamers of Cronobacter sakazakii were calculated by qPCR method.The results show that the Kd value of the original aptamer is 40.93 nM,and the Kd value of the optimized core sequence aptamer is 33.42 nM.It can be seen that the affinity has been significantly improved,which proves that our cutting optimization process is reasonable and the cutting is successful.However,it has been reported that the result of aptamer fitting determination of affinity is almost the same as the reported Kd value,which also proves the reliability of the qPCR method we chose.4.A highly sensitive fluorescence detection strategy for Cronobacter sakazakii without enzyme labeling was established.This method combines the advantages of enrichment ability of magnetic beads,efficient amplification of HCR,aptamer recognition of target,fluorescence signal accumulation of SYBR Green I and selective quenching of graphene oxide.It has high sensitivity and good specificity.The detection results showed that under the optimum conditions,the detection limit in the pure culture system was 2 CFU/mL,and there was a good linear relationship between the target concentration of 4.6～5×103 CFU/mL and 8 CFU/mL in the milk powder matrix,and the detection range was 1×101～1×103 CFU/mL.The comprehensive evaluation system of Cronobacter sakazakii and the aptamer tailoring process established in this paper provide new ideas for the selection and tailoring of other types of aptamers,which can be used for reference and practical value.