Research On Subtyping And Detection Of Diarrheagenic Escherichia Coli Based On Asymmetric Polymerase Chain Reaction Combined With Nucleic Acid Lateral Flow Assay

Diarrheagenic Escherichia coli（DEC）are common foodborne pathogens worldwide.Infected people tend to have diarrhea accompanied by vomiting,abdominal pain,fever,and other symptoms.In severe cases,kidney failure and even death can occur.DEC can be divided into five pathogenic types according to their pathogenesis.Rapid,accurate,and convenient subtyping and detection of DEC is essential for disease tracing and contamination control.Phenotyping methods are difficult to accurately detect DEC.The current national standard method（GB 4789.6-2016）uses polymerase chain reaction（PCR）-gel electrophoresis to identify characteristic gene fragments,but gel electrophoresis is complicated,time-consuming,and difficult to distinguish the amplification bands.The asymmetric PCR（a PCR）combined with nucleic acid lateral flow assay is an excellent method for the detection of DEC with its advantages of convenient operation,low cost,high detection speed,accuracy and high sensitivity.In the first chapter,the general situation of DEC,the methods of DEC subtyping,the detection techniques of molecular biology and immunochromatography were introduced.In the second chapter,a method for Enteroinvasive Escherichia coli（EIEC）detection and subtyping was developed by a PCR combined with nucleic acid lateral flow assay.Based on the characteristic genes of EIEC（inv E and uid A）,a PCR primers and oligonucleotide probes were designed,and then single strand DNA（ss DNA）products of uid A and inv E were prepared by a PCR,and spherical nucleic acid probes（SNAPs）for uid A and inv E were prepared using the salt-aging method.Hybridizing the nucleic acids labeled on two kinds of SNAPs with ss DNA mix products,EIEC subtyping was achieved with the nucleic acid lateral flow assay.The synthesis conditions of SNAPs and a PCR amplification conditions were optimized in the study.In the synthesis of SNAPs,the optimal amount of ss DNA modified sulfhydryl was 5×10^-4μmol,the optimal block agent was BSA,and the optimal amount of BSA was 0.9%（w/v）.In a PCR amplification,the optimal sense primer and antisense primers ratios of inv E and uid A fragments all were 1:3,the optimal concentration of primers were 0.25and 0.20μM respectively,and the optimal cycles was 40.The results showed that the proposed method was sensitive and could distinguish EIEC,E.coli and non-E.coli accurately with a detection limit of EIEC at 3.97×10^-3 ng genomic DNA.In the third chapter,a method for Enteropathogenic Escherichia coli（EPEC）detection and subtyping was developed by multiplex a PCR（m-a PCR）combined with nucleic acid lateral flow assay.Based on the characteristic genes esc V and uid A of EPEC,m-a PCR primers and oligonucleotide probes were designedand,then esc V and uid A ss DNA mix products were obtained by m-a PCR amplification,and SNAPs for esc V and uid A were prepared using the salt-aging method.Hybridizing the nucleic acids labeled on two kinds of SNAPs with ss DNA mix products,EPEC subtyping was achieved with the nucleic acid lateral flow assay.In the study,the amplification conditions of m-a PCR were optimized.The optimal ratios of sense primer and antisense primers for esc V fragment and uid A fragment were 1:5 and 1:3,the optimal concentration of primers were 0.25 and 0.2μM,the optimal cycles was 30,and the optimal annealing temperature was 58.2℃.The results showed that the proposed method had high sensitivity and could accurately distinguish EPEC,E.coli and non-E.coli with a detection limit of EPEC at 4.02×10^-4 ng genomic DNA.In the fourth chapter,the content of study was summarized,and the application of nucleic acid lateral flow assay in DEC subtyping and detection was prospected.In summary,a PCR combined with nucleic acid lateral flow assay and m-a PCR combined with nucleic acid lateral flow assay were established in this study to achieve accurate,sensitive,and fast subtyping detection of EIEC and EPEC,respectively.This study is conducive to early detection of related diseases caused by EIEC and EPEC,rapid identification of pollution sources,and important guarantee of food safety.