Application Of Hybrid Chain Reaction In The Detection Of Tylosin And Multiple Pathogens

Food safety,which is the basic guarantee of national safety,has received extensive attention and has been researched.The abuse of veterinary drugs and the contamination of microorganisms are important factors affecting food safety.Tylosin(TYL)is a macrolide antibiotic,mainly used for livestock,poultry breeding,and disease prevention and control.However,after long-term use,TYL is easy to accumulate in the human body and cause the resistance of pathogen.Therefore,it is necessary to establish a highly sensitive method for the detection of TYL.Food safety events caused by Escherichia coli O157:H7(E.coli O157:H7),Salmonella serotype Choleraesuis(S.Choleraesuis)and Listeria monocytogenes(L.monocytogenes)pose a great threat to human health.Therefore,it is important to establish a method that can simultaneously detect multiple pathogens.Hybridization chain reaction(HCR),as an efficient new amplification technique,can be proceed under the condition of constant temperature without the participation of enzymes.The highly sensitive and specific detection of TYL and the simultaneous detection of multiple pathogens based on HCR were achieved in this study.In the first chapter,the harm and the research progress of TYL and foodborne pathogens,and HCR are reviewed.In the second chapter,a method for detecting TYL by HCR combined with biotin-streptavidin signal amplification system was established.As an antibiotic,TYL promotes the development of animal husbandry,however,it poses a potential threat to human health.In this chapter,TYL monoclonal antibody(mAb)was prepared for immunological reactions,and the biotin-streptavidin system was combined with HCR to achieve double signal amplification.Streptavidin(SA)and mAb were labeled on gold nanooparticles(AuNPs)as probes(mAb-AuNPs-SA).In the absence of the target,the TYL-CMO-OVA-mAb-AuNPs-SA complex was formed through the immunological reaction.SA was combined with biotin-initiator.After adding the hairpins(H1 and H2),biotin-initiator caused the hairpins to open alternately for HCR.The fluorescent group modified on H1 was used as the signal output.The results showed that under the optimal conditions,the method had good specificity for the detection of TYL in PBS,the limit of detection(LOD)was 0.39 ng/mL,and it could be applied to the detection of TYL in milk and honey with LOD of 0.49 ng/mL and 0.25 ng/mL,respectively.In the third chapter,a multicolor ELISA platform based on fluorescent HCR was established to detect foodborne pathogens.Since pathogens are easy to cause human infection or poisoning,and multiple pathogens may coexist in the same sample,so it is necessary to establish a method that can specifically,sensitively,and simultaneously detect a variety of pathogens.In this chapter,three sets of hybridization sequences were designed,in which each set of initiators could only initiate matching H1 and H2.Each set of H1 was marked with non-interfering fluorescent groups.Enzyme-linked immunosorbent assay(ELISA)was combined with HCR to form an immune sandwich complex in the presence of target bacteria.Hairpins modified with fluorescent groups was added to trigger HCR,and a multicolor fluorescent cascatemers was formed as a signal amplifiers and signal reporters.The results showed that the platform could simultaneously detect E.coli O157:H7,S.Choleraesuis and L.monocytogenes,the LOD was 3.4 ×101,6.4 × 100 and 7.0 ×101 CFU/mL,respectively,with good specificity.Moreover,the platform could also be used to detect multiple pathogens in spiked milk samples.