Selection, Identification, Modification, And Application Of SsDNA Aptamers Against Vibrio Vulnificus

Vibrio vulnificus is a Gram-negative,halophilic,flagellated,ubiquitous,small size marine bacterium which can cause serious infection among people all over the world,especially those in coastal states and islands.V.vulnificus can be isolated from oysters and other marine mollusks,so people can be infected through contaminated foods.Being exposed to contaminated water with a preexisted cut is more easily to be infected.The mortality is high,and V.vulnificus is responisble for 82% of death caused by Vibrio bacteria.According to our national protocol,the detection of V.vulnificus is typically based on bacteria culture,so it may take 2-3 days to get accurate identification results.The onset time of V.vulnificus infection can be as fast as 4h,and if a patient did not receive any treatment in 72 h,he would be dead.Thus,rapid and sensitive detection methods for V.vulnificus are crucial for clinical diagnosis and food safety monitoring.In recent years,aptamers offer more choices for microbial diagnosis.Many aptamer-based biosensors were developed and widely used in diagnosis and food safety monitoring.Aptamers are ssDNA or ssRNA chains.Due to single-strand conformation polymorphism,nucleotide chains in same lengths can display different structures.That’s why aptamers are also called chemical antibodies.By incubating bacterium with same length,randomized ssDNA or ssRNA library,sequences that can specificifically bind to pathogens are enriched and selected.The selected aptamers can be easily produced and modified,and then combined with impedimetric biosensor,flow cytometry and colloidal gold detector to detect and identifiy all kinds of pathgens.By bacterium-SELEX,we obtained two aptamers with high binding affinity that can specifically bind to V.vulnificus ATCC 27562.When primer regions were removed,the random regions maintained their specificity and high binding affinity.Shorter sequences can lower cost of biosensors.The aptamers can recognize V.vulnificus in all culture status,and V.vulnificus in early exponential phase,late exponential phase,stationary phase and Viable But Non-Culturable status showed similar fluorescent intensity.Confocal fluorescence microscopic images showed that aptamers could pick V.vulnificus out from a mixture of various species of bacteria.When incubated in complicated environments such as proteinase K treated serum and oyster infusion,aptamer still showed good binding ability on V.vulnificus.The aptamers can not only recognize ATCC27562,but also other V.vulnificus strains,isolated from a patient and water,respectively.Though flow cytometry,we directly detect V.vulnificus in liquid samples by fluorescent-labeled aptamers.The test limit is 29.7CFU/mL,and the limit with accuracy is 100 CFU/mL.The linearity of this method is between 500-500,000CFU/mL.The test takes less than 1h,We also got bacteria debris by 20 min proteinase K treatment,and the fluorescent intensity on debris increased 3 times and the test limit can be as low as 7.804CFU/mL,and the limit with accuracy is 28 CFU/mL.Compared with traditional methods,our detection is faster and more sensitive.To conclude,we got V.vulnificus with high specificity and high binding affinity through bacterium-SELEX.Fluorescent-labeled aptamer made it possible to detect uncultured V.vulnificus more rapidly by flow cytometry.We can directly detect V.vulnificus in Viable but Nonculturable State more accurately.Aptamer-based biosensor can increase the efficiency of clinical diagnosis and food safety monitoring and have great potential to be widely used.