Establishment Of ELISA And PCR Diagnosis Methods For Trichinosis

Trichinosis is caused by Trichinella.It is a zoonotic disease that poses a serious threat to human health and is widely prevalent throughout the world.Infected persons have a higher infection rate due to ingestion of raw or unprocessed meat products containing infectious cysts.The main clinical manifestations of human infection are fever,dyspnea,diarrhea,and edema,which pose a great threat to human health.Trichinella excretion and secretion of antigens(ES antigens)are mainly produced by excretion and secretion of worms.Since the survival time of in vitro culture of Trichinella spiralis is short,ES antigens currently used in research mainly come from muscle larval stage.Because ES antigens can induce host immune responses,they are frequently used in the diagnosis,vaccines,and immunity of trichinellosis at this stage.Serine protease inhibitors(SPIs)are a class of protein superfamilies.Studies have confirmed that the presence of serine protease inhibitors in biological reactions such as coagulation and complement activation plays an important role.Trichinella spiralis is one of the few specific antigens of Trichinella spiralis ES antigen.It plays an important role in immunoprevention and diagnosis.Therefore,this study selected the Trichinella spiralis adulterase and Trichinella spiralis muscle larvae ES antigen for Trichinella spiralis.Establishment of an infection method.In this study,the purified serine protease inhibitors of Trichinella spiralis and the excretory and secretory antigens of Trichinella spiralis muscle were used as coating antigens to establish an indirect ELISA assay and a series of conditions were optimized.The optimal amount of TsAdSPI was determined to be 0.09375 ?g/well for the antigen and 1.5 ?g/well for the muscle larvae of Trichinella spiralis.The optimal serum dilution ratio was 1:400 for TsAdSPI and 1:100 for Trichinella spiralis muscle larvae.The optimal dilution ratio of secondary antibody was 1:6000 for TsAdSPI,1:5000 for T.spiralis muscle larvae,1:12800 for TsAdSPI,1:6400 for Trichinella spiralis muscle larvae,and TsAdSPI in the sensitivity test of the host.Infection with 20 Trichinella spiralis infections was positive at 14 days.ES antigens were detected as positive at the earliest days after infected with 20 Trichinella spiralis infections for 21 days.In specificity tests,Schistosoma japonicum and Clonorchis sinensis were used in this study.Cross-reactions occurred;replicates between batches and within batches were tested using batch plates with different batch numbers.The results showed that the coefficient of variation of both replicates was less than 10%.In 80 clinical samples,the positive rate of TsAdSPI was 36.25%(29/80)and the ES antigen was 26.25%(21/80).The test results showed that the sensitivity of TsAdSPI was higher than that of ES antigen.The purified TsAdSPI and Trichinella spiralis muscle larvae ES antigen were used for coating,and the NC membrane was used as the solid phase carrier to establish Dot-ELISA detection,and a series of conditions were optimized.The optimal amount of TsAdSPI was determined to be 0.25 ?g/tablet and the ES antigen was 2 ?g/tablet;the optimal serum dilution ratios were 1:400 and 1:200,respectively;the optimal secondary antibody dilution ratio was 1:8000,and 1:6000;Sensitive TsAdSPI can reach 1:12800,Trichinella spiralis muscle larvae ES antigen can reach 1:6400;In the host sensitivity test,TsAdSPI was detected as positive at the earliest infection of 50 Trichinella spiralis infections for 14 days,ES antigen was first infected 100 Trichinella spiralis infections were detected positive at 14 days;repeatability tests were performed on three batches of membranes and both proteins were reproducible.In the specificity test,Schistosoma japonicum and Clonorchis sinensis were used in this study.The results showed no cross reaction.The results of 80 clinical samples showed that the positive rate of TsAdSPI was 26.25%(21/80),and the positive rate of ES antigen of Trichinella spiralis was 15%(12/80).The test results showed that the serine proteases of adult Trichinella spiralis were inhibited.The agent is more sensitive than P53.Primers were designed and synthesized using TsAdSPI and P53 genes as target sequences.PCR was performed using DNA extracted from mice infected with different numbers of infected Trichinella spiralis strains for different days of infection.Positive and negative controls were set up,as well as adult serine proteases of Trichinella spiralis.The inhibitor PCR reaction program was denatured at 95°C.for 10 minutes,and was 30-35 cycles at 95°C.for 30 s,56.7°C.for 30 s,72°C.for 1 min,and 72°C.for 10 min.The P53 reaction program was denatured at 95°C.for 10 minutes,and 30-35 cycles at 95°C.for 30 seconds,52.9°C.for 30 seconds,72°C.for 74 seconds,and 72°C.for 10 minutes.After the PCR product was identified,the results showed that the target band was amplified by using the serine protease inhibitor of Trichinella spiralis as the earliest primer for the infection of 8 strains of T.spiralis for 24 hours.Specific samples were amplified in all test samples 7 days after infection.However,PCR was performed using P53 as a primer.The results showed that the target band could be amplified only after more than 50 infections with Trichinella spiralis and the infection time was 14 days.80 clinical specimens were detected using TsAdSPI primers with a positive detection rate of 43.75(35/80)and P53 primers with a positive detection rate of 13.75(11/80).Tests have shown that the adult Trichinella spiralis serine protease inhibitor is more sensitive than P53.