Investigation Of Enterotoxigenic Escherichia Coli F5 And Establishment Of Duplex RAA-LFD Detection Methods For F5 And F41

Enterotoxigenic Escherichia coli(ETEC)is a bacterial pathogen that causes calf diarrhea.Among them,ETEC F5 and F41 cause the most severe calf diarrhea disease.Understanding its epidemic situation and quickly and accurately diagnosing livestock infected with ETEC are the main measures to prevent the disease.Recombinase Aid Amplification(RAA)is an isothermal amplification technology.By combining with the Lateral Flow Dipstick(LFD),it can realize the visualization of nucleic acid detection.In view of the clinical detection and prevention and control needs of ETEC F5 and F41,this paper mainly conducts the following three aspects of research.1.Epidemic survey of Enterotoxigenic Escherichia coli F5 in large-scale cattle farms around HohhotA total of 404 manure and blood samples were randomly collected from 7large-scale cattle farms around Hohhot City,PCR and ELISA methods were used for detection.The results showed that 64 positive samples were detected by PCR in 404fecal samples,with a positive rate of 15.84%,and 135 of the 404 blood samples were positive for F5 by ELISA method,with a positive rate of 33.42%,among which the positive rate(17.82%)of calves aged 0～2 months was the highest.In summary,F5infection has existed in large-scale cattle farms in Hohhot,and the high incidence is in calves aged 0～2 months.2.Isolation and identification of Enterotoxigenic Escherichia coli F5 and pathogenicity researchBacterial isolation and culture were performed on PCR positive samples in flow modulation;Identification of isolates by molecular biology;Evaluation of drug susceptibility of F5 by susceptibility testing;A mouse model of diarrhea was constructed to study the pathogenicity of F5.The results showed that eight strains of F5 were identified by bacterial isolation.F5 isolates are resistant toβ-lactams and tetracyclines,and are sensitive to ofloxacin,imipenem and amikacin.Autopsy of mice infected with F5showed intestinal adhesion hematoma,liver and spleen enlargement and congestion;Analysis data showed that the half lethal dose(LD50)of F5 to mice was 10~7CFU/m L.3.Establishment of rapid detection method for duplex RAA-LFD of Enterotoxigenic Escherichia coli F5 and F41The F5 and F41 gene sequences were targeted,specific primers were designed,and a duplex RAA-LFD method capable of identifying Enterotoxigenic Escherichia coli F5and F41 was established.On this basis,the duplex primer and probe system were optimized,the sensitivity,specificity and clinical samples were tested,and the detection efficiency of the duplex RAA-LFD method was evaluated by PCR as a parallel comparison.The results showed that efficient nucleic acid amplification could be achieved by reacting at 39°C for 20 minutes,and plasmids as low as 2.5×10~3copies/μL could be detected.In addition,there was no cross reactivity with specificity testing.In clinical sample testing,the analysis of Kappa coefficient results shows that RAA-LFD is consistent with PCR methods.In summary,the duplex RAA-LFD detection method was successfully established in this trial to provide a new way for the clinical detection of ETEC F5 and F41.