In Vitro Selection Of DNA Aptamers And Fluorescence-based Recognition For Rapid Detection Of Listeria Monocytogenes

Objective: To screen high-affinity and high specificity aptamers to Listeria monocytogensby applying SELEX(Systematic evolution of ligands by exponential enrichment) method,and initially established a method for rapid detection Listeria monocytogens.Methods:1. The Listeria monocytogens as a target, a single stranded DNA random library with80nucleotides in length containing37random sequences was constructed. After ninerounds of SELEX selection, the high affinity and specificity of the aptamers with Listeriamonocytogens were obtained and the counter-selection against Bacillus cereus and Listeriainnocua were introduced in the5th and the7th rounds in order to improve the specificity ofthe screening. According to Digoxigenin-anti-digoxigenin-AP system we detected thebinding rate between the product every one round of selection and Listeria monocytogens.2. The last round of selection product were amplified, purified, cloned and sequenced.Application Chromas2software analysis aptamer sequencing peak diagram, applicationDNA MAN software analysis primary structure of homology, and RNA Structure softwareprediction secondary structure of sequence.3. Through the detection to aptamer of affinity and specificity, choose the best aaptamer. Using fluorescent combination mechanism to design two fluorescent aptamer,which used5’-FAM or5’-TAMRA labled. Initially established a rapid identificationListeria monocytogens method by fluorescence labled aptamers.Conclusions:1. Selection was more and more strict with the increase of the number of wheelscreening, the high affinity and specificity of the aptamers with Listeria monocytogens wereobtained. Preliminary established technology of in vitro selection of DNA aptamers forRapid detection Listeria monocytogens by SELEX.18clones were randomly selected to besequenced and the result was divided into3groups. Group A consisted of15fragments wasof the same random sequence as the expected fragment length, Group B consisted of2fragments was shorter than expected in the random region. Group C consisted of1fragments was increase1base sequence in the random region. Application DNA MAN software analysis primary structure of homology, all the sequence have high homologyexcept H09. This showed after nine rounds of SELEX selection, aptamers that have highhomology of primary structure got a certain degree enrichment. By RNA Structure softwareto predict the secondary structure, the result was of the main stem-loop structure.2. According to Digoxigenin-anti-digoxigenin-AP system we were detected thebinding rate of aptamers. We were selected the highest affinity of aptamer E01from theeighteen aptamers, and aptamer E01was combined with various strains for specificdetection. We found that E01aptamer and Listeria monocytogens was combined with ratethan other strains up to1.017，this showed aptamer E01high specificity.3. We were used5’-FAM and5’-TAMRA labled the optimal cloned aptamer E01, afteraptamer B10to directly bind with Listeria monocytogens, we could be directly observedstrong fluorescence by fluorescence microscope, while Bacillus cereus and Listeria innocuaonly a very small amount of the combination. Fluorescently labeled aptamer detected theensitivity of Listeria monocytogens was10~3/ml. Initially establish a rapid diagnosismethod by fluorophore-labled aptamers for the rapid detection of Listeria monocytogens.