Establishment Of XMAP Methods For Detecting Porcine Parvovirus,Porcine Reproductive And Respiratory Syndrome Virus,Porcine Circovirus Type 2 Antibodies

Reproductive disorders are the most important type of disease that causes pig production.Infectious diseases were important factors in causing reproductive disorders.Porcine Parvovirus(PPV),Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)and Porcine Circovirus Type 2(PCV2)were causative agents that caused reproductive failure and immunosuppressive disease in sows.Epidemiological investigations found that these three viruses were often mixed infected and made the disease worse.PPV causes reproductive failure of swine characterized by embryonic and fetal infection and death,usually in the absence of outward maternal clinical signs.PRRSV causes reproductive failure in breeding stock and respiratory tract illness in young pigs.PCV2 causes Postweaning Multisystemic Wasting Syndrome(PMWS).However,viral infection by itself tends to cause only mild disease,and co-factors such as other infections or immunostimulation seem necessary for development of severe disease.A definitive diagnosis of multiple infections is often difficult in that clinical signs can be variable and thus fail to provide a clear indication of the most appropriate means of testing.As a result,a number of costly individual,virus-specific tests are sometimes performed initially to expedite the testing procedure.Luminex x MAP technology is a bead-based multiplexed immunoassay system in a microplate format.The system can simultaneously detect many targets in a single sample,up to 500,depending on system design.The microspheres used in the Luminex system have different spectral addresses(color codes).These unique spectral addresses are created by internally labeling beads with different ratios of two fluorophores,one in a red wavelength and the other in the far red.Proteins specific to the desired targets can be coupled to the beads.As a new high-flux detection technology,x MAP can be applicable for the rapid serological detection of various pathogens,with high accuracy and simple operation that are superior to conventional methods.Multiple assays can be performed at the same time in asame well with less sample volume and other reagents.Internal assay control beads with test samples can be mixed in the same well to validate each test run,so less hazardous waste can be generated in the lab.In this study,the x MAP method to simultaneously detect PPV,PRRSV and PCV2 antibodies in serum was establisheds:PPV virus was used as antigen to establish a PPV x MAP method.The specific results showed no cross-reactivity with other virus positive serums,such as Classic Swine Fever Virus(CSFV),PCV2,Pseudorabies Virus(PRV),PRRSV.For comparison of x MAP with ELISA for PPV detection,29 serum samples were tested and the results shown the coincidence rate with the results of the ELISA method was 93.1%.Comparison to ELISA methods,the sensitivity of x MAP method to detect PPV was 16 times that of ELISA method.PRRSV N protein was used as antigen to establish a PRRSV x MAP method.The specific results showed no cross-reactivity with other virus positive serums,such as CSFV,PCV2,PRV,PPV.For comparison of x MAP with ELISA for PPV detection,29 serum samples were tested and the results shown the coincidence rate with the results of the ELISA method was 86.20%.Comparison to ELISA methods,the sensitivity of x MAP method to detect PRRSV was 16 times that of ELISA method.PCV2 Cap protein was used as antigen to establish a PCV2 x MAP method.The specific results showed no cross-reactivity with other virus positive serums,such as CSFV,PPV,PRV,PRRSV.For comparison of x MAP with ELISA for PPV detection,29 serum samples were tested and the results shown the coincidence rate with the results of the ELISA method was 62.07%.Comparison to ELISA methods,the sensitivity of x MAP method to detect PCV2 was 16 times that of ELISA method.Among 683 field samples,the detection results using Multiplex x MAP assay for PRRSV,PPV and PCV2 were 81%,86.68% and 69.98% consistent with those tested by conventional ELISA assay,demonstrated that the multiplex x MAP assay is high-throughput.The Luminex x MAP technology has many advantages over other methods of detecting and measuring antibody.Flexibility to singleplex or Multiplex,simultaneously measuring many targets in a single sample.Reduced cost and labor by multiplexing.Luminex assays have smaller sample requirements than other techniques such as ELISA.Comparison to ELISA methods,the sensitivity of x MAP method was better.In this study,the x MAP method was established to simultaneously detect PCV2,PRRSV and PPV antibodies in serum,optimized and applied for the differential diagnosis of PCV2,PRRSV and PPV and evaluating the effect of vaccine immunity by detecting PCV2,PRRSV and PPV antibody levels.