Establishment And Application Of Rapid Detection Method For Serotypes Of Avian Pathogenic Escherichia Coli O1,O2 And O78

Avian pathogenic Escherichia coli(APEC)is a type of enteropathogenic Escherichia coli(ExPEC),which can cause disease in poultry,including chickens,turkeys,ducks,geese,quails and pigeons.APEC can cause a variety of extraintestinal diseases in poultry,including respiratory infections,yolk sac infection,pericarditis,perihepatitis,air sacs,meningitis,head swelling syndrome,and sepsis.APEC is one of the important pathogens that endanger the health of the poultry farming industry.Many studies have shown that there is a close relationship between avian and human E.coli.They have high homology in genomic structure and high similarity in genetic evolution and ecological distribution,indicating that APEC is at risk of zoonosis.Therefore,the detection of APEC is of great significance.There are many O serotypes of E.coli,but the mainly serotypes that cause avian disease are O1 O2 and O78.In order to rapidly detect O1?O2?O78 serotypes of APEC,we established a triple PCR rapid detection method for O1,O2,O78 serotypes of APEC,which is suitable for rapid laboratory testing.In order to perform rapid screening on site,we originally wanted to prepare a colloidal gold immunochromatographic test strip for rapid detection of O78 serotype of APEC on site.However,the specificity of the obtained monoclonal antibodies is low,which is not suitable for the specific screening of O78 serotype E.coli.Therefore,colloidal gold nucleic acid chromatographic test strip was established for clinical rapid detection of O78 serotype of APEC,combining the PCR method with colloidal gold immunochromatography.1.Establishment and application of triple PCR detection method for O1,O2 and O78 serotypes of APECThree pairs of specific primers were designed according to the specific sequences of O1?O2 and O78 serotypes of APEC strains.The DNA of APEC O1?O2 and O78 serotypes strains were taken as templates.The reaction conditions of this triplex PCR were optimized.The optimal reaction condition is 25?L mixture,containing 1.5?L dNTP,2?L MgCl2 and 0.6?L Taq DNA polymerase.The optimal primer ratio was O1:O2:O78=0.6?L:0.2?L:0.6?L,and the optimal annealing temperature was 55.9?.The results of specificity test showed that there was no cross reaction between three serotypes.The specific detection of triple PCR by 146 different serotypes of E.coli showed that the accuracy of E.coli specific detection was 99.32%(145/146).There was no non-specific amplification when using Staphylococcus aureus,Salmonella and Bacillus subtilis for specific detection.Different concentrations of DNA were used for sensitivity detection of triple PCR.When single serotype of DNA as used as template,the minimum detectable concentrate of DNA for O1 and O2 serotypes was both 0.1 ng/?L,for O78 serotype DNA was 0.02 ng/?L.When mixed DNA of O1?O2 and O78 serotypes was taken as template,the minimum detectable concentrate was 0.05 ng/?L.18 clinical simulation samples were tested with a positive rate of 94.4%(17/18).2.Monoclonal antibody preparation of O78 serotype of APECIn order to establish a colloidal gold immunochromatographic method which can used to rapidly detect APEC in the clinic,a monoclonal antibody against O78 serotype of APEC was prepared in this study.Based on the specific gene of O78 serotype of APEC,a pair of specific primers containing NdeI and XbaI restriction enzyme cutting sites were designed,and the target fragment was amplified.The recombinant plasmid pCznl-AGC87681.1 was constructed by restriction enzyme digestion and ligation,and transformed into Arctic Express(DE3).The O78 protein was expressed by IPTG hypothermia induction.Since protein was expressed in the inclusion body,we first performed protein renaturation and then purified the protein.BALB/c mice were immunized with purified O78 protein.After cell fusion,screening and subcloning,two positive monoclonal cell lines were obtained,named 2D3 and 8B4.The titer of monoclonal antibody reached 1:512000.However,there is a significant cross-reaction between different serotypes of E.coli strains,so it is not suitable for the preparation of colloidal gold immunochromatographic test strips to detect O78 serotype of APEC.In the next step,a specific polypeptide can be screened by the phage peptide library based on the obtained monoclonal antibody to establish other detection methods.3.Development and application of APEC O78 serotype nucleic acid colloidal gold test stripIn order to rapidly detect O78 serotype of APEC in clinic,we combined the PCR and colloidal gold immunochromatography technique to prepare a colloidal gold nucleic acid chromatographic strip.Primers were designed based on the specific fragment of O78 serotype APEC.The forward and reverse primers were modified with FITC and DIG,respectively.Then PCR products with double labeling of FITC and DIG were obtained by amplification,which was used to be tested by colloidal gold nucleic acid chromatography strips.A colloidal gold solution with a particle size of 15-20nm was prepared by trisodium citrate reduction method.We prepared gold standard antibody by adding 4?L of lmg/mL FITC antibody to 1mL colloidal gold solution and using 0.5%gelatin as blocking solution.The T-line and C-line on the NC membrane were respectively labeled with digoxin antibody and goat anti-mouse secondary antibody.The sensitivity of the prepared colloidal gold nucleic acid chromatographic test strip was 102CFU/mL.17 strains of non-O78 serotypes of E.coli were used to test the specificity of the strips and the result showed that there was no lines appeared on the T-line,indicating the high specificity of the colloidal gold nucleic acid chromatographic test strips.Different concentrations of O78 serotype of APEC strains were added to the serum as simulate samples for detection.The results showed that the limit detection was 102CFU/mL.With this colloidal gold nucleic acid chromatography strip,rapid and on-site detection of O78 serotype APEC can be achieved without any expensive instrumentation or professionalism.