Establishment And Application Of PCV2 Fluorescence Quantitative PCR Detection

Since the discovery of porcine circovirus 2（PCV2）in Canada in the early 1990s,PCV2has become one of the most important infectious agents of swine,and is the main pathogen of multisystem failure syndrome（PMWS）,porcine dermatitis nephrotic syndrome（PDNS）and congenital tremor of piglets（CT）.PCV2 can seriously damage the immune system of pigs,resulting in a variety of diseases,resulting in increased mortality,decreased feed conversion rate,low production performance of pigs and other adverse consequences.In order to understand the detoxification of PCV2 in large-scale pig farms and reduce the loss caused by PCV2 virus,it is necessary to establish a rapid,accurate and efficient qPCR detection method,which can be used for pathogen monitoring of PCV2 in pig farms and evaluation of commercial vaccine effect,and provide reference for selecting suitable PCV2vaccine and formulating reasonable immunization program for pig farms.In this study,a pair of probes and primers were designed according to the PCV2 ORF2gene of PCV2 virus in GenBank.The PCV2 positive samples were selected for amplification of the target gene by ordinary PCR,gel cutting recovery,TA cloning,positive cloning screening and identification,etc.The target gene was successfully cloned into p MD18 vector to obtain positive recombinant plasmid.The obtained plasmids were diluted by 10-fold gradient.qPCR was used to conduct sensitivity test and construct standard curves for 7gradients 10^-4-10^-10,and the intra-batch and inter-batch repeatability tests were carried out.The results showed that the standard curve equation was CT=-3.1761lg X+38.977,and the correlation coefficient R²=0.9943.The minimum detection limit was 6.56×10⁰copies/μL,with coefficients of variation less than 5.0%in and between batches.The established qPCR kit and commercial PCV2 qPCR kit were used to detect 40 clinical samples.38 positive samples were detected by the established qPCR kit in this study,and 37 positive samples were detected by commercial kit.The qPCR method established in this study has better detection effect than the commercial kit of the same kind,and can be used for monitoring PCV2 in large-scale pig farms.The established qPCR was used to evaluate the immune effect of vaccines,and the result showed that:In the comparative test of clinical antiviral immunity effect of commercial PCV2 vaccine,the pathogen positive rate and overall detoxification volume of pigs in each group were calculated.At 130 days of age,the pathogen positive rate of pigs in group C（vaccine C）was 80%,and the overall detoxification volume of pigs in group C was371.46 copies lower than that in other experimental groups and the control group.Group C had the best clinical antiviral effect.In the effect comparison test of PCV2 vaccine with different times of immunization,the mean values of viremia,oral and nasal detoxification volume and fecal detoxification volume of pigs in each group were calculated,and the viremia of pigs in group H（the first immunization group）,group G（the second immunization group）and group F（the third immunization group）were 237.28 copies,112.28 copies and 164.72 copies.Group H had the lowest detoxification volume,with4088.27 copies of oral and nasal detoxification volume and 3522.61 copies of fecal detoxification volume of pigs in this group.Considering the viemia,oral and nasal detoxification volume and fecal detoxification volume of pigs in each group,group H had the best immune effect.qPCR detection effect of own method is superior to the similar commercial kits,followed by the commercialization of PCV2 vaccine clinical antiviral immune effect of contrast test and PCV2 vaccine immunity to the effects of the number of comparative tests,the vaccine C immune effect is best,the immunization effect of vaccine C was the best,and the difference of viremia was little in different immunization times,and the detoxification amount of group H was the lowest,It is suggested that vaccine C should be the first choice in actual production,and vaccine C could get a better effect,which piglets was immuned at 14days of age.