| Clonorchis sinensis is a serious hazard of freshwater zoonosis of parasitic diseases. Infection for people or mammals mainly occurs by taking in raw or undercooked freshwater fish. It can eventually lead to gallstone, cholangitis, hepatolithiasis, cholangiocarcinoma and liver cirrhosis in chronic infection. Metacercariae to mature adults start eject of eggs with fece when after 4 weeks in infection of clonorchis sinensis, if the inspection found no eggs in the fece will lead to misdiagnosis during this time, and delay in the treatment of diseases, it may lead to serious consequences. Therefore, diagnosis of clonorchiasis has a great significance to preventing this disease.Firstly, this research applied stool test(modified kato thick smear method) and has published PCR detection method to obtain standard positive serum in clonorchiasis epidemic region. Stool examination of 1745 people, and 513 of them have eggs in feces; then the PCR identification of 513 DNA that extracted in stool samples, result shown positive for 113 people, obtain positive serum of 112. This research through the double identification which can reduce the false positive serum, determine obtain the standard positive serum of clonorchiasis is 112, and using this serum detection sensitivity of indirect ELISA diagnostic method.This study intend to use clonorchiasis diagnosis method based on immunology principle in view of rapid development for the immunological diagnostic methods in diagnosis. Currently, ELISA diagnostic method has been gradually applied to the diagnosis of clonorchiasis, using this method can carry on the diagnosis of disease as soon as possible, it not only can avoid delay in the treatment of diseases, but also can solve the blind spot problem for diagnosis of infection in the early and in period of eggs can’t produce. Diagnosis antigens applied for Indirect ELISA diagnosis method almost was secretion of crude antigens or ES antigens, although sensitivity of these antigens can be high, but specificity is poor, besides there are false positive in healthy people, meanwhile its cross reaction is very strong with other parasites such as worms, opisthorchis fluke, and colonization. Currently, gene engineering antigens has becoming a research hot in diagnostic antigens, its on the basis of high sensitive, at the same time has high specificity, also can solve the problem of cross reaction. Therefore, looking for high specificity, high sensitivity of diagnosis antigens will become the key of immunological diagnostic methods.In order to solve above problem, early in our laboratory screening with clonorchis sinensis adult c DNA expression library by immunology, obtain 3 strong reaction antigens, and the sequencing of three antigens are highly repeat sequences, and all have the same end, therefore, this experiment design for four pairs of antigen gene according to the three strong reaction antigens of repeat sequence and common terminal, respectively is Cs G1, Cs G2, Cs G6, Cs Gend, first synthesis gene of four antigens,and four antigens for prokaryotic expression, successful preparation of expression vector p ET-28a-Cs G1、p ET-28a- Cs G2、p ET-28a-Cs G6、p ET-28a-Cs Gend,ultimately obtain four gene recombinant antigens, the purity above 95%. Western blot results show that the four kinds of recombinant protein could be specificity identification to positive serum. Using this four recombinant antigens respectively to establish clonorchiasis indirect ELISA diagnostic method. The sensitivity of four recombinant antigen respectively was 91.1%(102/112)、86.6%(97/112)、74.1%(83/112)、 49.1%(55/112), the sensitivity of four antigens together package was 83.9%(94/112); All specificity is 100%. Cs Gend what the common end of three strong reaction of antigenic genes from Sieve repository which antigenicity is weakest; Cs G1 antigenicity is strongest, it is the ideal diagnostic antigens,using this antigen to establish indirect ELISA diagnostic method of clonorchiasis, it will be can solve the problem of early infection cannot be diagnosed, and has advantage to prevention and control of clonorchiasis. |
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