Screening The Aptamers Of Ustiloxin A And Constructing Visualized Nucleic Acid Probe

Rice false smut is one of the most important rice fungal diseases in the world,which seriously threatens crop yield and food safety,and also spreads easily in the field by spore transmission.Therefore,early diagnosis is crucial for effective control of this fungal disease.Ustiloxins are produced during the occurrence and development of rice false smut,and Ustiloxin A is the dominant in the early stage.The highly sensitive Ustiloxin A detection may be an effective way to achieve rice false smut early diagnosis.Nucleic acid aptamers have been developed into an important class of detection and diagnostic tools,due to their specific binding capability.SELEX screening technology is the most important way to obtain the aptamers of targets,and a large number of aptamers(including various aptamers of mycotoxins)have been successfully screened using this method.Using aptamers as signal detection elements,we can develop visual nucleic acid sensors based on RCA,which greatly amplify detection signals through DNA amplification.Screening the aptamers of Ustiloxin A and then designing visually ultrasensitive nucleic acid sensor,have great significance for the rice smut disease early diagnosis.We have carried out systematic research in the following two aspects.1.Using high-purity Ustiloxin A,we obtained the target binding motifs using magnetic beads-based SELEX by 14 rounds of positive,negative and reverse(using glutathione,glycine,and Ustiloxin D)selections.Through in-depth analysis,we found that Ustiloxin A had preference to bind DNAs containing GATGG,GAGACCATC,and CTCAGATG units.Based on the motif sequences,we designed P1-P3 DNA sequences and verified that P1-P3 could bind to Ustiloxin A.The binding ability of Ustiloxin A to P1-P3 increased with the number of screening rounds.2.Using PDGF-BB and its aptamer as a model,we designed a CDNA that can competitively bind to the aptamer to initiate RCA amplification.The RCA products could bind to urease-conjugated DNA and then catalyze the hydrolysis of urea into ammonia,which made the phenol red indicator turn red.After optimization,the sensor had less background interference and achieved highly sensitive detection of the target PDGF-BB.For example,12 pmol PDGF-BB significantly turned the base solution form yellow to red.The sensor was also specific for the detection of PDGF-BB,and BSA or IgG could not discolor the base solution.In summary,we successfully obtained DNA motifs binding with Ustiloxin A by Mag-SELEX,and designed a RCA-based visual nucleic acid sensor.Our work has reference significances for both designing visual nucleic acid sensors of Ustiloxin A and developing early diagnosis tools of rice smut disease.