The Study Of Selection, Structural Analysis And Specificity Of Aptamers To Listeria Monocytogenes

In this study, in order to lay a foundation for the detection of Listeria monocytogenes in food, SELEX method was utilized to screen DNA aptamers that specially bind Listeria monocytogenes with a high affinity.In vitro selection process against Listeria monocytogenes, reverse screening method was used to remove the common antigen that exited the bacteria to accelerate the process of aptamer enrichment against specific antigen on the bacteria. After 14 rounds of selection, the screened products were cloned and sequenced. The primary structure and secondary structure of the sequenced aptamers was analyzed. The combination rate and specificity of the aptamers was also studied.The SELEX screening results showed that the combination rate of ssDNA enriched library of each round and Listeria monocytogenes had increased, and the absorbance value had raised nearly 2.5 times, from 0.264 of the first round to 0.659 of the fourteenth round. The conserved sequences of primary structure also revealed that five pairs of aptamers had identical sequences. There was only one different base in sequences of another part of aptamers. And 11 aptamers had the sequence of CCC(TG)C(G)GGG(CA). This suggested that the aptamers with primary structure homology had been enriched to some extent. The secondary structure consisted mainly of Stem-loop structure.This implicated that these Stem-loops were the basis for aptamers binding to target molecules as well as the basis for its further form the three-dimensional structure. The experiments of aptamer binding to Listeria monocytogenes showed that the combination rate of NO. B09 aptamer was maximum, and the combination rate of NO.127 aptamer was minimum. The specificity experiments of NO. B09 aptamer revealed that the absorbance value of NO. B09 aptamer binding to Listeria monocytogenes was maximum (0.760). This implicated that NO.B09 aptamer had a certain degree of specificity. And this had laid a certain foundation for subsequent applied research.