Establishment Of Indirect ELISA Serological Detection Of Enterococcus Faecium

Enterococcus faecium,as a main pathogen of the hospital epidemic,is an important conditional pathogens for humans and animals.It Mainly causes urinary tract infection,abdominal and pelvic infection and sepsis,etc.,and causing zoonoses.The lack of rapid and accurate clinical detection methods for Enterococcus faecium limits the epidemic control of the disease.Therefore,the establishment of a fast,simple,specific and high sensitive diagnostic kit has an potential economic importance.In this study,the methods of microscopic examination,PCR amplification and sequencing,mouse virulence test,virulence gene and drug resistance gene amplification experiment were used.A strain of Enterococcus faecium,named temporarily Enterococcus faecium B1 Y,was isolated from a moribund sheep in Sunan,Gansu province.The strain has a strong pathogenicity and multiple drug resistance.The sequence of the 16 S rRNA gene was 100% similar to Enterococcus faecium strain ISMMS_VRE₁1 and Enterococcus faecium isolate 2014-VREF-268 in GenBank.We decided to establish indirect ELISA serological detection of Enterococcus faecium based on the Enterococcus faecium B1 Y strains.Capsular polysaccharide?CPS?,an important virulence factor,is an important hapten,and immunogenicity can be enhanced coupled with corresponding protein.In order to extract and purify CPS and determine its polysaccharide content,the methods of high pressure method and enzymatic digest were used.After ultrafiltration concentration and DEAE SEPHAROSE FAST FLOW column chromatography,The purified CPS was obtained.The content of the purified polysaccharide was 75.21% determined by phenol-sulfuric acid method.To enhance the immunogenicity and specificity of CPS,the CPS was successfully conjugated with BSA.The conjugated protein with high immunogenicity was used as an antigen to establish the method of ELISA.The results showed that the P/N values were greater than 1,can significantly distinguish between negative and positive serum.But the negative values were large,the optimum condition still need to be optimized.Adhesin of collagen from E.faecium?Acm?is an important virulence factor of Enterococcus faecium,which has the effect of adhering the intestinal tract and preventing it from being cleared.In this study,the Acm gene was amplified by PCR and cloned to pUC57-Kan vector.The results showed that the gene was 1514 bp in total,encoding about 501 amino acid residues,and the Acm sequence had 99% similar to the published Acm of E.faecium by aligned with the sequence in Gen Bank.The prokaryotic expression plasmid pET-32a-Acm was constructed by clone the Acm gene into the pET-32a?+?vector by BamH I and Xho I digestion,and was transferred into E.coli BL21?DE3?to induce its expression.SDS-PAGE and Western blot analysis showed that the recombinant soluble protein was obtained and the molecular weight of the protein was about 74 ku,which could react specifically with the positive sera of E.faecium and had a good reactionogenicity.Concentration of the target protein purified by His Ni affinity chromatography column was 1.2 mg /mL.The purified Acm protein was coated and the indirect ELISA detection method for E.faecium was established.The optimum conditions for the optimal screening were as follows: The optimal conditions for ELISA were as follows: the concentration of recombinant antigen was 12 ?g / m L,and then at 4 ?overnight after incubation at 37 for 2 h,10% horse sera was selected as blocking solution and 37 ? ?for 1.5 h.Diluted serum samples to be tested for 1:100,37 ?1 h incubation.he optimal dilution of HRP was 1:500,the optimum working condition was 37 for 15 min,The substrate color time was 37 ? ?for 5 min.On the basis of this,the cutoff value was 0.427 by determined with 24 negative sera.73 samples of sheep serum were detected with indirect ELISA method and compared to nucleic acid amplification assay and sequencing?NAAAS?.The positive coincidence rate of the indirect ELISA was 63.64%?7/11?and the negative coincidence rate of 87.10%?54/62?,and the total coincidence rate was 83.56%?61/73?when compared with nucleic acid amplification assay and sequencing.Established ELISA method was of high specificity,good repeatability and higher sensitivity.