Creation And Evaluation Of Aptamer-based Novel Methods For Detection And Control Of The Selected Pathogenic Microorganisms

Salmonella is the most common bacterial pathogen that causes foodborne diseases.Rapid and accurate detection of the pathogenic bacteria in food and clinical samples is crucial for a timely control of Salmonella infection.Although traditional methods for salmonella detection are still proposed nowadays,they have various limitations.Currently,much attention has been paid to the development of novel detection methods that are simple,reliable,inexpensive,rapid and efficient.Another pathogenic microorganism that seriously threat human health is influenza virus.It causes influenza,one of the most serious life-threating infectious diseases.Nowadays the treatment of influenza still relies on the conventional M2 ion channel blockers and the neuraminidase inhibitors.However,with the rapid variation of influenza virus and the ever-growing number of drug-resistant strains,the efficacy of conventional drugs becomes more and more compromised.Therefore,it is important and urgent to identify and develop antiviral drugs with new mechanisms of action against influenza virus.In view of these problems and considerations,we tried to establish an aptamer-based platform for rapid and efficient detection or control of several kinds of pathogens that pose great threats to human health.Focusing on such topic,we have carried out the following aspects of work in this study:1.Screening of aptamers targeting Salmonella typhimurium via cell-SELEX procedure.By using Salmonella typhimurium(S.typhimurium)as selection target,6 DNA aptamers with high specificity and strong binding capacity were selected via the cell-SELEX procedure.All their dissociation constant(Kd)values were lower than 100 nM,of which the Kd value of StmApt 6 was 30±4 nM,the lowest one,representing that StmApt 6 had the highest binding affinity for its target.Also,StmApt 6 showed a good ability of discrimination between S.typhimurium and other types of bacteria,such as Salmonella choleraesuis(S.choleraesuis),Salmonella paratyphi A,Staphylococcus aureus(S.aureus),and Escherichia Coli K88(E.Coli K88).2.Screening of aptamers targeting Salmonella paratyphi A flagellin and H1N1 influenza virus hemagglutinin with standardized SELEX method.By using the purified Salmonella paratyphi A(S.paratyphi A)flagellin and H1N1 virus hemagglutinin(HA)proteins as selection targets,4 DNA aptamers targeting the flagellin and 2 DNA aptamers against the HA with high affinity and specificity were isolated through the standardized SELEX procedure.All their Kd values were lower than 100 nM,of which the Kd values of FlagApt 3(aptamer to flagellin)and HaApt 1(aptamer to HA)were the lowest,as 27±27 nM and 78±1 nM,respectively.3.Construction of S.typhimurium detection system by using the selected nucleic acid aptamer and graphene oxide.A nanomaterial-and 5'-fluoro-aptamer-based detection aptasensor was built by modifying the above-mentioned StmApt 6 with FAM at the 5' end,followed by a non-covalent binding to graphene oxide.This aptasensor allowed a rapid and effective detection of&typhimurium,with an excellent sensitivity of detectable limit being as low as 100 CFU/mL,and a satisfactory linear correlation ranging from 103 CFU/mL to 108 CFU/mL.The performance of this system in the case of real sample detection was also tested by using milk spiked with S.typhimurium as a real sample.The result showed that the system had a good reactivity to the target bacteria in milk,suggesting that the aptasensor is of significant practical application value in the qualitative and quantitative detections of S.typhimurium in food.4.Construction of S.paratyphi A detection system by using the selected nucleic acid aptamer and single-walled carbon nanotube.A dual signal detection aptasensor was constructed by modifying a truncated form of the above-mentioned FlagApt 3 with FITC at the 5'end,and covalently attaching a DNA sequence whose G-quadruplex-hemin complex possesses a peroxidase-like activity to the 3' end,followed by a non-covalent binding to single-walled carbon nanotube.The amount of S.paratyphi A in a sample could be measured by determining either the fluorescence intensity or absorbance when a sample was added into the system.The aptasensor was applied to the quantitative determination of S.paratyphi A spiked into milk samples,and the target bacteria were detected by 105 CFU/mL(fluorophotometry)and 106 CFU/mL(spectrophotometry).As dual signals are measured simultaneously for detection of the same target bacterium,this method can significantly reduce the possibility of false positive rate,and thus greatly improve the accuracy and reliability of the detection results.5.Study on the antiviral effect of the selected nucleic acid aptamer.The antiviral activities of the above-mentioned HaApt 1 aptamer were studied by hemagglutination inhibition test and microneutralization assay.The results showed that HaApt 1 significantly inhibited the erythrocyte aggregation induced by H1N1(A/Puerto Rico/8/1934)influenza virus,and prevented the viral infection and replication in the cultured MDCK cells.Based on these results,a preliminary judgment could be made that HaApt 1 may be tightly bound to the critical site of the H1N1 HA protein,thus blocks the interactions between the virus and host cell receptors.Therefore,HaApt 1 may be a competent candidate for detection of the critical binding site of HA protein,and hence possesses potential application values in the study on the mechanisms of its antiviral action and development of H1N1-specific virucidal agents.