Detection Technology Of Porcine Circovirus Type 2 And Development Of Diagnostic Kits

Porcine circovirus type 2(PCV2) causes Post weaning multisystemic wasting syndrome(PMWS),Pocine respiratory disease complex(PRDC),and porcine dermatitis-nephrotic syndrome(Porcine dermatitis and nephrotic syndrome,PDNS)and other diseases have caused great economic losses to the global pig industry.Various diagnostic techniques such as Polymerase chain reaction(PCR),Immunohistochemistry(IHC),Immunofluorescence assay(IFA),In situ hybridization(In situ hybridization),ISH)has been used for the detection of PCV2 antigen or antibody.However,these methods require specialized testing equipment and a high level of expertise,and are only suitable for laboratories.This study started from the virology and pathogenesis of PCV2,obtained recombinant protein by prokaryotic expression and prepared polyclonal antibody.On the basis of purified recombinant proteins and polyclonal antibodies,ELISA antibody detection kits,latex immunochromatography antigen detection test strips and magnetic bead chemiluminescence antigen detection kits are developed.These three kits greatly improve the detection capacity,shorten the detection time and simplify the detection process under the premise of ensuring good sensitivity,specificity and stability.1、The ELISA antibody detection method for qualitative analysis is established with the purified recombinant Cap protein as the coating antigen.The optimization results of process parameters showed that the optimal antigen coating concentration was4 μg/m L.The optimal blocking was 2% of BSA.The optimal blocking conditions were4℃ for 16 h.The optimal serum dilution was 100 times.The optimal dilution of enzyme-labeled antibody is 10,000.The optimal serum incubation time is 30 min.The optimal enzyme-labeled antibody incubation time is 30 min.The optimal TMB time is10 min.The experimental results of the performance verification show that the sample still has good sensitivity under the condition of 1600-fold dilution.Positive results only are found in the detection of PCV2 antibody-positive serum,and the detection results of other virus antibody-positive sera are all negative results.PRRS,PRV,PPV,PEDV,CSF antibody-positive serum has no cross-reaction.The intra-assay coefficient of variation is 1.4%,and the inter-assay coefficient of variation is 2.46%,with high precision.The rate of clinical samples is 98%.The kit can be stored for 12 months at 4℃with good stability.2、Anti-Cap protein polyclonal antibodies and latex microspheres were used to prepare immunoprobes.The anti-Cap protein monoclonal antibodies and goat anti-rabbit Ig G secondary antibodies were used to prepare T line and C line,respectively.The optimization results of process parameters showed that the optimal diameter of latex microspheres was 300 nm.The optimal binding amount of polyclonal antibody was 80 μg.The optimal spraying amount of immunoprobe was 1.5 μL/cm.The best C-line point film concentration was 1.0 mg/m L.The experimental results of performance verification show that the detection limit of the test strip is 0.5 ng/m L.What is more,it only shows positive results in the detection of PCV2 antigen-positive serum samples,and it has no cross-reaction with antigen-positive serum samples of other porcine viruses.It has good sensitivity and Specificity.The comprehensive detection rate of clinical samples is 93.33%.It can be stored for 12 months at room temperature.3、Immunoprobes are composed by combining polyclonal antibodies and magnetic beads microspheres.luminescent probes are composed by monocloning antibody and acridinium ester.The optimization results of process parameters showed that the optimal feeding ratio was 1:10.The optimal binding amount of polyclonal antibody was68 μg.The optimal binding time was 120 min.The optimal immunoprobe blocking was2% of BSA.The optimal amount of immunoprobe was 100 μL.The optimal dilute proportion of sample was 100.The optimal dilute proportion of secondary antibody was4000.The optimal sample incubation time was 25 min.The results of the performance verification show that the kit has good linearity in the antigen concentration range of0.25 ng/m L to 250 ng/m L.The detection limit is 51 pg/m L,and the quantification limit is 68 pg/m L,the kits has a high Sensitivity.The coefficient of variation between batches is less than 5%.The comprehensive detection rate of clinical samples is 97.14%.The kit can be stored at 4℃ for at least 12 months.