| Porcine viral infectious diseases are rapid and widespread infectious diseases in swine diseases.They are characterized by rapid spread,widespread epidemics,and mixed infections,which can cause serious clinical symptoms such as severe immunosuppression and reproductive disorders.Bring huge economic losses.Classical swine fever（CSF）,Porcine circovirus type 2（PCV2）,Porcine Reproductive and Respiratory Syndrome（PRRS）The industry has brought huge economic losses and seriously affected the development of animal husbandry.Therefore,the establishment of an accurate and rapid diagnostic method is extremely important for the pig industry and public health related to the pig industry.The liquid phase chip detection method is a new biochip technology developed by Luminex Corporation of the United States through the combination of gene chip technology and flow cytometry technology.The technology innovatively uses fluorescently encoded microspheres as a reaction carrier,and the microspheres are suspended in a liquid phase environment to provide a reaction condition close to a natural state for the reaction of biomacromolecules.The aim of this study was to establish a rapid,reproducible,highly specific and highly accurate single-fluorescence microsphere immunological detection method for porcine viral disease IgG antibody,and to establish a scientific integration of IgG antibody multiplex fluorescence in three diseases.Microsphere immunological detection method.Firstly,p MAL-c5x-CSFV-E2,p MAL-c5x-PCV2-Cap and p ET-32a-PRRSV-NSP7recombinant plasmids were constructed using p MAL-c5x and p ET-32a prokaryotic expression vectors.CSFV-E2、PCV2-Cap fusion MBP and His-tagged recombinant proteins and PRRSV-NSP7 fusion His-tagged recombinant proteins were obtained.The recombinant protein was analyzed by SDS-PAGE and Western blot.Purified by Ni2+column affinity chromatography,the recombinant protein with good immunogenicity and antigenicity was obtained,which laid a prerequisite for the establishment of further FMIA method.The CSFV-E2,PCV2-Cap and PRRSV-NSP7 recombinant proteins were coupled as antigens to the 60,40,and 10 beads microspheres to obtain E2-60,Cap-40,and NSP7-10coupling complexes.Its capture vector was established to detect CSFV-E2,PCV2-Cap and PRRSV-NSP7 IgG antibodies by FMIA.When tested by a single method,the minimum detection concentrations for CSFV,PCV2,and PRRSV monoclonal antibodies are respectively 1ng/m L,CSFV,PCV2,PRRSV serum dilution reached Mmax（Median Fluorescent Intensity）at 1:100,1:50,1:200,respectively;no cross-reactivity with other disease-positive sera common in pigs;CSFV,PCV2 The average intra-assay coefficient of variation of PRRSV single IgG antibody fluorescent microsphere immunoassay method was7.6%,6.4%,and 3.2%,respectively,and the average inter-assay coefficient of variation was6.9%,6.7%,and 4.2%,respectively.The results of clinical serum samples were analyzed by ROC(r-eceiver operating characteristic（ROC）curve.The sensitivity of CSFV,PCV2,PRRSV single IgG antibody fluorescent microsphere immunoassay was 98.9%,99.0%,94.9%,respectively.The traits were 96.8%,91.3%,and 91.1%,respectively.When the cut-off values were 5939,2882.5,and 5991.5,both the liquid protein chip assay and the ELISA could reflect the same index.Based on a single FMIA,the CSFV,PCV2,and PRRSV microsphere coupling complexes were placed in the same reaction system,and a triple FMIA method for simultaneous detection of CSFV,PCV2,PRRSV antibodies was established and validated,and a single detection method was established.The test results are linearly correlated and can reflect the same indicator.In summary,this study successfully constructed p MAl-c5x-CSFV-E2,p MAL-c5x-PCV2-Cap and p ET-32a-PRRSV-NSP7 recombinant plasmids by prokaryotic expression vectors p MAL-c5x and p ET-32a.Soluble recombinant protein,which is coupled with microspheres as antigen,establishes a single FMIA detection method for CSFV,PCV2 and PRRSV IgG antibodies,and establishes multiple FMIA detection methods.The establishment of this method can lay a foundation for further establishment of multiple detection methods for swine virus disease antibodies,and provide ideas for the establishment of new serological diagnostic techniques for other animal disease antibodies. |
