| With the development of society economy and urbanization in our country,the number of pets has been increasing day by day.Pet zoonosis incidence is also increasing,due to the increasing variety of pet diseases,the pathogenic mutation also accelerated.The changes of people’s and pets’ lifestyle can promote the prevalence of pet zoonosis.Enterotoxigenic Escherichia coli(ETEC),Yersinia enterocolitica(Y.enterocolitica)and Campylobacter jejuni(C.jejuni)are the most important bacterial disease,they can cause diarrhea in both human beings and other animals.Currently,many methods can be used to detect these three bacteria.These methods meet the requirements in terms of specificity,sensitivity,and detection limits.However,they still have some limitations,such as time-consuming processes,more reagents used,complex operation,propensity for false positives,low efficiency and other shortcomings.They are difficult to meet the requirements of rapid testing.Thus,developing novel approaches for the rapid detections of antibodies against pathogenic ETEC,Y.enterocolitica and C.jejuni in serum has great significance.Fluorescence Polarization assay(FPA)is a homogeneous detection system,it has the characteristics of simple,reliable,quick and efficient.It can meet the development trend of high throughput,high speed,multi-residue and automation.Nowadays,FPA have been successfully applied to many research fields,including high-throughput screening of drugs or clinical disease diagnosis.In this study,the specific protein of enterotoxigenic ETEC,Y.enterocolitica and C.jejuni were respectively labled by fluorescein.New fluorescence polarization assays were established with the labeled product and achieve simple,sensitive and rapid detection effect.The main contents and results are as follows:Ⅰ.The FaeG of ETEC,ail of Y.enterocolitica and Peb1 A of C.jejuni are expressed by prokaryotic expression,then identified the recombinant proteins and indicated that our recombinant proteins had satisfied immunogenicity.Ⅱ.Under alkaline conditions,FITC reacted with the amino group of a protein to form a FITC-protein conjugate.Then it was characterized by spectrophotometry and agarose electrophoresis.Ⅲ.Polyclonal antibodies were prepared respectively by artificial infection of rabbits with ETEC,Y.enterocolitica and C.jejuni.The polyclonal antibody titers were detected by indirect ELISA,which were 1:64 000,1: 128 000 and 1: 102 400.Ⅳ.The tracer which had been confirmed the optimal diluted concentration and reaction time,then we respectively established the FP assay for detection of antibody to ETEC,Y.enterocolitica and C.jejuni.Its specificity,sensitivity,repeatability were tested.Results are as follows:i.The FP assay method for detection of ETEC antibody had a minimal detection titer of 1:80,this assay only reacted with ETEC positive serum and had no cross-reactivity with other serum,it had high specificity.The coefficients of variation,both inter-and intra-assay,were less than 10%,it has good repeatability.ii.The results showed that the FP assay method for detection of Y.enterocolitica antibody had a minimal detection titer of 1:120,this assay only reacted with Y.enterocolitica positive serum and had no cross-reactivity with other serum,it had high specificity.The coefficients of variation,both inter-and intra-assay,were less than 10%,it has good repeatability.iii.The FP assay method for detection of C.jejuni antibody had a minimal detection titer of 1:160,this assay only reacted with C.jejuni positive serum and had no cross-reactivity with other serum,it had high specificity.The coefficients of variation,both inter-and intra-assay,were less than 10%,it has good repeatability.Ⅴ.Using our FPA method and antibody ELISA kit to detect two hundred serums,the results indicated that the total coincidence rate of ETEC was 95%,total coincidence rate of Y.enterocolitica was 93%,total coincidence rate of C.jejuni was 89%.In conclusion,the gene of ETEC FaeG,Y.enterocolitica ail and C.jejuni Peb1 A are expressed by prokaryotic expression,FITC respectively marked protein to form a tracer.We respectively established the FP assay for detection of antibody to ETEC,Y.enterocolitica and C.jejuni.Then the results indicated that this method has high specificity,sensitivity,repeatability and coincidence rate.They meet the simple,sensitive and rapid detection requirements,which can be applied to the rapid detection of antibodies in serum and provide technologies and products for the epidemiological investigation and diagnosis of bacterial diseases in pets. |
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