Aptamer Screening And Establishment Of An Aptamer-based Method For Detection Of The Food-borne Bacillus Cereus And Shigella

Both Bacillus cereus and Shigella are two common food-borne pathogens,which can cause dizziness,diarrhea,vomiting and other diseases.They are typical representative strains of gram-positive bacteria and gram-negative bacteria,which can usually be detected in unqualified milk and meat products.Therefore,establishment of a rapid and accurate detection method is of great significance for food safety and people's healthy life.At present,the traditional detection methods used at home and abroad are subjective and time-consuming.The preparation cycle of antibodies for ELISA is long and antibody is difficult to transport and preserve.Molecular biological methods are prone to false positive because of environmental interference.Hence,establishing a detection method that combines the advantages of all parties has important research value.We established an aptamer captured system combined with fluorescence quantitative PCR(qPCR)to detect the two foodborne pathogens.This study includes the following researches:(1)Screening of aptamers of Bacillus cereus and Shigella:Using Bacillus cereus and Shigella as the targets to be detected.,an artificial 80 bp length ssDNA library containing each of 20 bp fixed sequence at both ends,and a 40 bp random sequence in the middle of it.After ten rounds of positive selections and two counter selections,two types of aptamers with high specificity and affinity were obtained by improved SELEX technology.In order to improve affinity,incubation time was shortened and washing times were increased with the rounds increased.The binding rate of aptamers to Bacillus cereus and Shigella was respectively determined to be 0.922 and 0.842 by the color reaction of digoxin alkaline phosphatase.(2)Cloning and sequence analysis of two kinds of aptamers:20 aptamers of Bacillus cereus and 17 aptamers of Shigella were obtained by amplification,cloning and sequencing of the products of the final round selection.We used DNAMAN software to analyze the homology and the secondary structure and predict free energy of aptamers.The results showed that the homology of aptamers was not high but the accumulation of the same bases was common.The secondary structures included stem rings,hairpins and bulges,which formed abundant spatial structures.It is concluded that secondary structure is the basis of specific binding between aptamer and bacteria.(3)Specificity analysis of aptamers of Bacillus cereus or Shigella:The specifity of aptamers obtained by sequencing were analyzed by fluorescence microscopy.The aptamers with strong binding ability and high specificity were selected for the establishment of subsequent detection methods.Firstly,the plasmids containing aptamers were amplified by FAM-labeled upstream primers and biotin-labeled downstream primers.Nextly,the single-stranded DNA aptamers labeled by FAM were isolated by magnetic bead method and incubated with target and non-target bacteria.The fluorescence signal was observed under fluorescence microscope.The results showed that the binding ability and specificity of aptamer L4.2 to Bacillus cereus and that of aptamer B13 to Shigella was strong and specific.(4)Establishment of an aptamer-qPCR methodsfor detection of Bacillus cereus or Shigella:The research in this section included synthesis of biotin-labeled aptamers L4.2and B13,establishment of aptamer capture target bacteria combined with qPCR to detect pathogenic bacteria,and application of this method in artificially contaminated bulk milk.The results showed that the sensitivity of L4.2 capture system combined with qPCR for detection of Bacillus cereus could reach 5.0×10~1 CFU/mL.The Cycle threshold(Ct)detected by qPCR had a good linear relationship with the concentration logarithm of Bacillus cereus solution,the linear equation is y=-3.5477x+35.89,and the correlation coefficient was 0.9983.The sensitivity of detection of Shigella by B13 capture system combined with qPCR was 3.0×10~1 CFU/mL.There was a good linear relationship between the detection of Ct value of qPCR and the concentration logarithm of Shigella solution.The linear equation is y=-3.353x+38.963,and correlation coefficient was0.9961.Using the established method to detect artificially contaminated Bacillus cereus and Shigella in bulk milk,the recovery rates were between 80 and 120%.In this study,the modified SELEX technique was used to screen aptamers that specifically bind to Bacillus cereus or Shigella.Based on the aptamers selected,a method of aptamer capture combined with qPCR for detection of pathogenic bacteria was established.The method has good specificity and sensitivity.The aptamers can be synthesized quickly,preserved and transported more easily than antibodies.A kit for rapid detection of pathogenic bacteria may be developed based on the method.The method provides a new way for detecting foodborne pathogens and a new idea for food safety detection.