| Porcine Circovirus Disease is caused by Porcine Circovirus Type 2(PCV2)infection.Its clinical symptoms are multiple system failure syndrome of weaned piglets,dermatitis nephrotic syndrome and reproductive disorders of sows.It is one of the important diseases that seriously endanger the healthy development of the pig industry and has caused huge economic losses to the pig industry around the world.The research on rapid diagnosis and detection technology of the disease has been a hot topic for scholars in various countries.Although diagnostic and detection techniques such as polymerase chain reaction(PCR),enzyme linked immunosorbent assay(ELISA),direct immunofluorescence,virus isolation and identification have been established,but there is a lack of technical methods to quickly read the detected values.Therefore,the research on the rapid reading and detection of PCV2 antigen antibody is necessary for the successful prevention and control of the disease.Based on prokaryotic expression and purification of cap protein,a rapid detection technique of PCV2 antigen fluorescent microsphere immunochromatography was established.Based on the paired PCV2 monoclonal antibody,a rapid detection technique of PCV2 antibody fluorescent microsphere immunochromatography was established,enriching PCV2.Rapid detection technology for antigen and antibody.In this study,702 bp cap gene was successfully amplified,prokaryotic expression vector of pET30a(+)-cap was constructed,and cap fusion protein containing his tag with molecular weight of about 36 Ku was expressed.Through the optimization of conditions,the optimal conditions for protein induction and expression were determined.The rotation speed was 180 rpm,the IPTG induction final concentration was 0.5 mM,the induction time was 5 h,and the induction temperature was 37?.Cap fusion protein was successfully purified by affinity chromatography.The purified protein concentration was 1.5 mg/mL.Western blot analysis showed that the purified protein can react with PCV2 specific polyclonal antibody and hasgood reactivity.A rapid imunochromatographic assay method for detection of porcine circovirus type 2antibody fluorescent microspheres was established by expressing purified cap fusion protein labeled fluorescent microspheres containing His tag.The unlabeled cap protein and rabbit-derived PCV2 high serum-free IgG were coated on the detection line(T)and quality control line(C)of nitrocellulose membrane respectively,and the parameters of membrane spotter were set to Speed 30 mm/sec and 1.0 uL/cm.The final concentration of fluorescent microsphere labeled protein antigen is 50 ?g/mL.The optimal reaction condition is to dilute the serum sample to be detected 20 times.Add 3 ?L of fluorescent microsphere coupled with antigen into 100 ?L of sample,incubate for 5 min after mixing evenly,add sample adding hole,start timing when the sample liquid completely infiltrates NC membrane,react for 15 min,read T/C ratio with dry immunofluorescence analyzer,and determine the result.After testing 340 serum samples from 8 different regions,compared with the commercial porcine circovirus ELISA antibody detection kit,the specific coincidence rate was 94.2%,the sensitivity coincidence rate was 99.63%,and the overall coincidence rate was 98.53.%;The test results show that the established rapid detection method of PCV2 antibody fluorescent microsphere immunochromatography is fast,simple,sensitive and specific,and can be used for the rapid detection of clinical porcine circovirus type 2 antibody.The PCV2 antigen fluorescent microsphere immunochromatographic assay was established based on two PCV2-specific monoclonal antibodies 5C and 4E11 based on the double-antibody sandwich method.Fluorescent microspheres were labeled with 5C monoclonal antibody,4E11 monoclonal antibody of 1mg/mL and goat anti-mouse IgG of1mg/mL were coated on the detection line(T)and the quality control line(C),respectively.The parameters of the dot film analyzer were set to Speed 40mm/sec,1.0 ?L/cm,and the final concentration of antibody fluorescent microsphere coupling was 150 ?g/mL.The best conditions for detection are as follows: the sample containing PCV2 is diluted 20 times,monoclonal antibody coupled fluorescent microspheres are added to every 100 ?L of the sample,mixed evenly in the dilution plate,incubated at room temperature for 5 min,added into the sample adding hole,timing is started when the sample liquid completely infiltrates the NC membrane,the reaction lasts for 12 min,and finally the T/C ratio is read by a dryimmunofluorescence analyzer to judge the result.120 samples collected and prepared in the clinic were tested.Compared with the commercial PCV2 PCR test kit,the specific coincidence rate was 97.22%,the sensitivity coincidence rate was 100%,and the overall coincidence rate was 99.17%.The results showed that The rapid detection method of PCV2 antigen fluorescent microsphere immunochromatography has the characteristics of rapid,simple,sensitive and specific,and can be used for rapid detection of clinical samples of PCV2 and rapid detection and quality control of semi-finished products in PCV2 vaccine production. |
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