| Clonorchis sinensis,an important foodborne parasite,mainly lives in the hepatic duct of mammals such as dogs,cats and humans causing clonorchiasis.Globally,approximately 35 million people are suffering from clonorchiasis,primarily in East and Southeast Asia,with an estimated 13 million infected people in China alone,and prevalent in 27 provinces especially Heilongjiang,Jilin,Guangdong and Guangxi.Hence,the prevention and control of clonorchiasis is urgent.The traditional etiological examination,which include microscopy for eggs and autopsy for worms,remain the diagnostic gold standard.Though technically specific,it suffers from low sensitivity,among which,the lightly infected cases can be missed,moreover,due to morphological similarities,the eggs of C.sinensis are easily confused with other flukes.Therefore,it is necessary to establish a more rapid,accurate,simple diagnostic method suitable for large-scale clinical sample detection.This experiment mainly includes three parts.(1)Collection of C.sinensis metacercaria and preparation of standard serum.The pure metacercaria was collected from naturally infected Pseudorasbora parva.Japanese white rabbits and Chinese garden dogs were infected orally with metacercariae,and the C.sinensis standard positive serum was prepared.(2)Analysis,cloning and expression of diagnostic antigen and screening of the optimal diagnostic antigen.Four diagnostic potential proteins,16-Ku calcium binding protein(cbp16),dynein light chain-2(DLC2),glutathione transfer ASE omega-2(gst-2)and paramyosin(PC2),were selected based on the bioinformatics analysis.Then the proteins were expressed and purified for Western blot reaction.The results showed that all of the four proteins can react with C.sinensis positive serum but not react with negative serum,which confirmed that these proteins had good diagnostic value.The four proteins were coated at ELISA plate with 5μg/m L,respectively,incubated with negative and positive serum at the same time.The results showed that,DLC2 protein showed the highest P/N value reaching4.13,indicating that DLC2 protein was the optimal antigen for the diagnosis of clonorchiasis.(3)Preparation and preliminary application of immunochromatographic test strip for rapid diagnosis of clonorchiasis.The detection line was crossed with DLC2 protein at the concentration of 0.5 mg/m L,the colloidal gold was coupled with spa as a marker,and the quality control line was crossed with 1 mg/m L chicken anti spa secondary antibody.The method of colloidal gold immunochromatography(GICA)test strip was established,and the dilution release times of rabbit,dog and human serum were optimized respectively.The established GICA method has no cross reaction with the positive sera of Schistosoma japonicum,Fasciola hepatica and Echinococcus japonicus,which has good specificity and can be used for rapid diagnosis of clinical samples.The detection results of clinical samples showed that the positive rate of pet hospital samples was 2.1%(5/138),and the stray dogs was5.6%(5/88).In this study,the C.sinensis infection model of dogs was successfully established,and a large number of standard C.sinensis positive sera were obtained.Four antigens with diagnostic potential were successfully expressed and purified,among which,DLC2 protein was selected as the antigen by ELIS with the largest P/N value,and an immunochromatographic test strip was established for rapid diagnosis of clonorchiasis.The GICA detection method showed a higher sensitivity compared with traditional feces egg test,and has a similar sensitivity and specificity to commercial human clonorchiasis kit,which provides a new method for the clinical diagnosis of clonorchiasis. |
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