| Fasciola hepatica（F.hepatica）is an important zoonotic parasite,can cause significant economic losses to animal husbandries,especially the sheep industry.After being infected by F.hepatica leads to anemia and jaundice,and threatens the health in humans.Therefore,the detection of F.hepatica is particularly important.At present,the conventional detection methods of F.hepatica mainly include etiology examination,molecular biological detection and immunological detection.With the advantages of good stability,high sensitivity and specificity,immunological detection is widely used.The principle of this method is antigen-antibody binding reaction,so this method can detect unknown antigen or antibody based on the known antibody（or antigen）to achieve the purpose of diagnosis.This method makes up for the shortcomings and deficiencies of traditional diagnostic methods.Although some specific genes,such as cysteine protease and cathepsin L,were used to detect F.hepatica,the number of diagnostic gene is limited.In this study,we aimed at exploring novel diagnostic gene by screening the c DNA expression library of adult F.hepatica.The recombinant candidate antigens of Fasciola hepatica were obtained and used to establish indirect ELISA and colloidal gold technology for F.hepatica infection.Then these two methods were used to detect clinical samples from sheep to evaluate the potential of this diagnostic gene This study may provide new candidate antigen for the detection of F.hepatica infection and technical support for the prevention and treatment of fascioliasis to achieve the goal of ensuring the healthy development of animal husbandry.Screening,expression and purification of candidate diagnostic antigen genes for.hepatica Adult F.hepatica c DNA expression library was used to screen novel diagnostic gene by SEREX technology.12 candidate diagnostic antigen genes of F.hepatica were obtained.Two of these genes has been reported previously,and ten of them were newly discovered candidate diagnostic antigen genes.Then F.hepatica.amoebapore-like gene in these newly discovered genes was successfully expressed,and F.hepatica.amoebapore-like protein（AYA57790.1,abbreviated as FHA5）was obtained.Establishment and preliminary application of an indirect ELISA method based on the diagnostic antigen FHA5 for detection of F.hepatica infection.An indirect ELISA detection method for Fasciola hepatica infection was established by using recombinant FHA5 protein.After the optimization of conditions,the best antigen coating volume of this method was 0.187μg/well,the best dilution of the sample was1:800,the best blocking agent was 5%skimmed milk powder,the best dilution of HRP-labeled rabbit anti-goat antibody was 1:5000;the best reaction time for the substrate was 20 min.The sensitivity can reach 1:12800,and it had no cross-reactivity with Dicrocoelium-positive serum.Faeces samples and serum samples from 36 sheep in Jilin were respectively detected by fecal examination and this indirect ELISA method.Result showed that 24 and 26 positive samples were found by fecal examination and the indirect ELISA method,respectively.And the positive rate of F.hepatica infection was 66.7%（24/36）and 72.2%（26/36）by these two methods,the coincidence rate of examinations of these two methods was 92%,the indirect ELISA method had a higher sensitivity.Then,a total of 80 serum samples isolated from sheep in Heilongjiang were detected by both commercial ELISA kit and our indirect ELISA method.The same result was detected by the both methods,39 samples were positive,and the positive rate was 48.7%（39/80）.These data indicated that FHA5 used in the indirect ELISA may be a candidate of diagnostic antigen for detecting F.hepatica infection.Establishment and preliminary application of the colloidal gold strip for F.hepatica infection Colloidal gold strip was made by using the recombinant FHA5protein of Fasciola hepatica.After the optimization of conditions,the optimal p H value was the p H value when 2μL 0.2 mol/L K2CO3 was added,the optimal labeling amount of SPA antibody was 6μg,the optimal coating concentration of the detection line（T）was 0.5 mg/m L,the best coating concentration of the quality control line（C）was 0.125 mg/m L,the sensitivity of this method can reach 1:160;there was no cross-reaction with Dicrocoelium-positive serum,these indicated that this strip had high sensitivity and specificity.The repeatability test and Preservation test showed that the storage times of this strip under the conditions of 4℃,room temperature and37℃were 90,60 and 60 days,respectively,this method showed good repeatability and stability.A total of 80 clinical serum samples isolated from sheep in Heilongjiang were detected using the colloidal gold strip,results showed that positive samples were39,and the positive rate was 48.7%（39/80）.Result of this strip was same with that detected by our indirect ELISA method,it indicated that this colloidal gold strip also had high sensitivity and stability.In summary,a candidate diagnostic antigen gene FHA5 was obtained by screening adult F.hepatica c DNA expression library in this study,used to establish an indirect ELISA method and a colloidal gold strip for detecting F.hepatica infection.After the preliminary application,these two methods showed high sensitivity and specificity.This study provides a novel candidate diagnostic antigen for the detection and prevention of F.hepatica infection. |
