Establishment And Application Of PCR-ELISA For Mycoplasma Suis

Mycoplasma suis is a kind of zoonosis,which is parasitic on the surface,plasma and bone marrow of the pig erythrocyte,which causes fever,jaundice and anemia of unknown cause.Because of the more and more serious damage caused by the animal infection of the erythrocyte,the disease has attracted the attention of all countries,especially in recent years,it has caused great loss to the livestock breeding industry in our country.At present,the disease has no standard immune control methods and special therapeutic drugs.Therefore,it is of great significance to establish a detection method suitable for rapid diagnosis of the disease,and to reduce the health hazards of the disease to human and animal,and to control and prevent the disease.In this study,a pair of specific primers were designed and synthesized according to the Eno gene sequence of the Mycoplasma suis published by GenBank.The 5 'end of the primers were labeled with biotin(BIOT)and digoxin(DIG),and the PCR products were amplified.According to the PCR-ELISA method,the optimal conditions were determined by optimizing the variety of the envelope,the concentration of the inclusion fluid,the reaction conditions of the antibody,the sealing condition,the dilution of the PCR product and the color time.The critical value of the PCR-ELISA method established in this study was calculated by measuring the OD values of 30 negative samples.The sensitivity of the method was detected by detecting the OD value of gradient DNA dilution of the genome of porcine erythrocytes.The specificity of the established PCE-ELISA method was detected by amplification of Mycoplasma suis,Streptococcus suis,swine Escherichia coli,Mycoplasma porcine mycoplasma,Toxoplasma gondii and healthy pig blood gene DNA.The stability of the PCR-ELISA method was tested by repeatability test of the same batch and batch batches of enzyme labeled plates.The PCR-ELISA method was applied to detect 40 pig blood samples from pig farms in Yanbian area and compared with the PCR-ELISA method established by Xia Xiaohui.The results showed that the size of the conventional PCR was about 170bp,which was consistent with the expected results.The PCR-ELISA optimization results showed that the best reaction conditions such as the buffer solution of pH 9.6,the concentration of the chain enzyme Vidin,the concentration of the chain enzyme avidin,the enzyme standard two anti selective 1:2 000,the 3%BSA sealing solution,the 2 mg/mL salmon sperm DNA,the closing time 60 min,the 30 times dilution of the PCR product and the chromogenic 15 min of the substrate.The critical value of PCR-ELISA method established in this study is 0.192.The lowest concentration of genomic DNA detected by porcine erythrocytes was 0.33pg,and the sensitivity was 10 times higher than that of traditional PCR method.The PCR method and the established PCR-ELISA method did not detect the PCR products of Streptococcus suis,swine Escherichia coli,Mycoplasma porcine mycoplasma,Toxoplasma gondii,which showed that the method had good specificity.The coefficient of variation of intra batch repeatability test and inter batch repeatability test was lower than 10%,which proved that the method was stable.The results of clinical samples showed that the positive rate was 22.5%,indicating that the sensitivity of the method was further improved.This study established a sensitive,specific and safe detection method for Mycoplasma suis,which is of great significance for the early prevention and timely control of Mycoplasma suis in pigs.