Establishment And Preliminary Application Of A Mutiplex PCR For Four Swine Reproductive Failure Viral Diseases

ObjectiveEstablish multiple PCR detection method that can be applied for clinical detection simultaneously of Porcine reproductive barrier disease,which is caused by Porcine parvovirus virus(PPV),Porcine circovirus type 2(PCV2),Pseudorabies virus(PRV),European type porcine reproductive and respiratory syndrome(EU-PRRSV)and American type porcine reproductive and respiratory syndrome(NA-PRRSV),and perform for preliminarily clinical application.MethodsWe used Oligo6.0 to design five pairs of specific primers in reference to the complete conservative genome sequences of PPV ? PRV ? PCV2 ?EU-PRRSV and NA-PRRSV in Gen Bank.Their products were 142bp,547bp,705 bp,1367bp and 331bp.On the basis of single PCR,we established multiple PCR detection method.We optimized the multiple PCR reaction conditions and tested specificity,sensitivity and repeatability.And we performed parallel test of part of the clinical samples and literature method to verify the reliability of the method.We performed application test for 325 samples from 5 cities in western Liaoning with the established multiple PCR detection method.ResultsIn the method of multiple PCR detection the suitable primer concentration of PPV ? PRV ? PCV2 ? NA-PRRSV and EU-PRRSV were 1.0?mol/L,0.5?mol/L,0.5?mol/L,1.5?mol/L and 1.5?mol/L.The annealing temperature was 60?.The minimum testing nucleic acid content were 155.0pg?45.2pg?17.2pg?14.2pg and 43.6pg respectively,which all reached the level of pg.We repeated testing the same samples at different time for 6 times and got the same results.We did parallel test with the literature method,and the coincidence rate was 100%.We tested clinically collected 325 samples of suspected porcine disease materials using multiple PCR detection method,the results showed that PPV,PRV,PCV2,NA-PRRSV and EU-PRRSV infection rate were 21.23%?53.53%?57.23%?52.00% and 2.77% respectively.ConclusionsThe investigation establishs multiple PCR detection method of PPV,PRV,PCV2,EU-PRRSV and NA-PRRSV,and the method has the advantages of rapidity,accuracy and sensitivity,which can be used in clinical diagnosis.