Immuno-HCR Signal Amplification Detection For Escherichia Coli O157:H7 And Logic Codes Immunochromatographic High-Throughput Serogrou Ping Assay For Salmonella

Escherichia coli O157:H7 and Salmonella are common food-borne pathogens.The infection dose of Escherichia coli O157:H7 is as low as 10 bacteria.It is necessary to sensitively detect Escherichia coli O157:H7.Salmonella have numerous serogroups and have difficults in source tracing for Salmonella.Therefore,it is necessary to establish a rapid and high-throughput method for Salmonella serogrouping.The sensitive detection for Escherichia coli O157:H7 and high-throughput serogrouping method for Salmonella were established in the study.Hybridization chain reaction(HCR)is an enzyme-free isothermal nucleic acid self-assembly reaction.In recent years,HCR has been widely used in the study of signal amplification.In the second chapter,an immuno-HCR was established by combining immunology and HCR to detect Escherichia coli O157:H7.When initiator was co-labeled with antibodies on a latex nanoparticles(LNPs),antibodies would interfere with initiator's reactivity in HCR,resulting in weak positive signals and low sensitivity.A complementary oligonucleotide(C-Initiator)was designed to labeled with antibodies on a kind of LNPs.Initiator was labeled on another kind of LNPs.The strategy reduced interference of antibody to Initiator in HCR reaction.Compared with immuno-HCR based on single LNPs(2,7×10~3 CFU/mL),the sensitivity of immuno-HCR based on dual LNPs(4.8×10~2 CFU/mL)increased 5.6 times.Salmonella is a common pathogenic bacterium.Salmonella isolates have been sorted into different serogroups according to the specific lipopolysaccharide present in the somatic surface(O antigen)of the bacterium.The virulence and pathogenicity of Salmonella vary greatly between different serogroups.Salmonella of O:2,O:3,O:4,O:7,and O:9 serogroups contribute to a large share of Salmonella outbreaks.Traditional slide agglutination tests to classify Salmonella serogroups are tedious because of their low testing efficiency,complex operation,and subjective judging criteria.Immunochromatography have been extensively described in the literature,which discussed their low cost,rapid detection,and portability in multitude of fields,such as agriculture and environmental sciences.In the third chapter,an immune-codes serogrouping assay based on immunochromatography was designed for rapid and high-throughput classification of five Salmonella serogroups.The five tests for the serogroups(i.e.,O:2,O:3,O:4,O:7,and O:9)were integrated in codes by three test lines.The sensitivity and specificity for the five serogroups exceeded 96%and 97%,respectively.No expensive or large instruments were needed in the immune-codes serogrouping assay.With these advantages,the proposed method deserves to be promoted and expanded.