| With the large-scale and intensive development of pig farming in China,the epidemic diseases associated to porcine reproductive failure have a trend of increasing incidence during pig farming,causing significant economic losses.And there are a variety of common pathogens associated with porcine reproductive disorders,porcine circovirus type 2(PCV2),porcine circovirus type 3(PCV3),porcine parvovirus(PPV)and pseudorabies virus(PRV)often lead to similar clinical symptoms and mixed infections to different degrees in swine herd.How to detect and identify these four DNA viruses quickly and accurately is one of the primary challenges to be solved in this study.In this study,two sets of primers and Taq Man probe combinations required for fluorescence quantitative PCR were designed among the conserved regions of PCV2 cap gene,PCV3 cap,PPV NS1 and PRV g E genes,which have already been deposited in Gen Bank for simultaneous detection and identification of these four pathogens,specific primers,probes and standard,plasmids were screened via verifications of the specificity,sensitivity and repeatability.Finally,an efficient,rapid and accurate multiplex Real-time PCR method was established,which began to be applied in clinical practice.After screening specific primers and probes,the designed multiplex Real-time PCR method has an excellent sensitivity:the minimum detection copy of PCV2 genome was 1 copy/μL,and the minimum detection copies of PCV3,PPV and PRV was 10 copies/μL.The correlation coefficients(R~2)were greater than 0.997 for different target genes.Genomic c DNA samples of porcine reproductive respiratory syndrome virus(PRRSV),porcine epidemic diarrhea virus(PEDV)and classical swine fever virus(CSFV)were used as templates for specificity evaluation.The results showed that the established method had an excellent specificity and no cross-reaction with genomic c DNA of other RNA viruses.Repeatability tests(>3 times)were performed on different samples.The results showed that the established method had an excellent repeatability.The coefficient of variation for the four target genes was less than 1%,and the coefficient of variation between groups was less than 3%.By test multiple samples with different types of instruments,the difference of Ct values obtained is less than 2,indicating that the established method has a good instrument applicability.Further,315 clinical samples from Hunan Provincial Center for Animal Disease Control and Prevention and laboratory collection were used to evaluate the clinical application of the optimized multiplex Real-time PCR method.The results showed that the positive rates of PCV2,PCV3,PPV and PRV was 66.67%(210/315)and,8.57%(27/315),8.89%(28/315)and4.13%(13/315),respectively.There were 39 samples with two kinds of mixed infections,and the mixed infection rate was 12.70%.In the mixed infections,19 samples were mixed with PCV2 and PPV,and 4 samples were mixed with three or more pathogens.In conclusion,the multiplex Real-time PCR method established in this study has high specificity and sensitivity,which can effectively identify four viruses PCV2,PCV3,PPV and PRV simultaneously,and can be applied to the clinical detection and diagnosis of the pathogens of the pathogens associated with porcine reproductive disorders. |
