Study On Detection Of Vibrio Alginolyticus And Vibrio Harveyi Based On Their Aptamers

Vibrio alginolyticus and Vibrio harveyi are the common conditional pathogens in aquaculture environment.They are widely distributed in ocean and some estuary watersheds.The two highly pathogenic vibrios can infect lots of aquatic animals,and bring huge economic losses to the aquaculture industry around the world.Therefore,rapid detection and monitoring of V.alginolyticus and V.harveyi are the necessary basis for prevention and control of the diseases caused by the two Vibrios.Aptamers are nucleic acid molecules with high affinity and specificity,exhibiting great advantages and application potential in the detection of microorganisms.In the present dissertation,the affinity and specificity of aptamers targeting at V.alginolyticus,V.harveyi and their inactivated form were studied,and the affinity constants of the aptamers were determined.The six aptamers(#7,#9,#37,I22,S27,S35)were proved to have good affinity and specificity toward their targeting bacteria,and their affinity constants were13.56±5.01,34.55±11.34,40.28±13.39,33.42±13.24,26.21±8.73,and 29.42±7.91 nM,respectively.Aptamer#37 against V.alginolyticus,and aptamer S35,and S27 against V.harveyi,had weaker affinities with the none-targeting bacteria(Aeromonas hydrophila,Escherichia coli,and Edwardsiella tarda),which show better affinity and specificity toward their targeting bacteria.The secondary structures of aptamers were also simulated and analyzed based on the base pairing with high fidelity.Study on the qualitative detection indicates that aptamers#7 can detect more than 10~2CFU/m L of inactivated V.alginolyticus,aptamers#9 and#37 can detect more than10~2 and more than 10~3 CFU/mL of V.alginolyticus,aptamer I22 can detect more than 10~2 CFU/m L of inactivated V.harveyi,and both aptamers S27 and S35 can detect more than 10~2 CFU/mL of V.harveyi.The qualitative detections of V.alginolyticus and V.harveyi based on aptamers were established preliminarily.Study on the quantitative detections of the two Vibrios indicate that the standard curves of aptamers#9 and I22 had better linear fitting.The quantitative detection based on aptamers is easy,efficient,and highly specific,does not require the extraction and purification of bacterial genes and PCR amplification,but its stability and accuracy still need to be imporved.