Molecular Epidemiology Investigation Of PCV2 And PCV3 In Hebei Province And Establishment Of Double Nano PCR Detection Method

Circovirus disease is an organ dysfunction or reproductive disorder caused by circovirus infection.The disease is recognized worldwide as one of the major diseases that seriously harm the pig industry.When mixed with other viruses,it is often Pigs cause serious harm.In order to understand the epidemic characteristics and genetic evolution of PCV2 in Hebei Province,this study established a PCV2 and PCV3 dual nano-PCR detection method.379 clinical samples from pig farms in Hebei Province were tested for PCV2 and PCV3,and 47 positive samples were tested for PCV2 ORF2 gene sequencing analysis,partial PCV2 and PCV3 full gene sequence analysis,the results are as follows:1.The PCR method was used to detect 379 clinical samples.The results showed that the positive infection rate of PCV2 reached 53.6%,the positive infection rate of PCV3 was lower at 25.6%,and the mixed infection rate of PCV2and PCV3 was 12.1%,indicating that PCV2 and PCV3 infections are more common in northern parts of our province.The genetic evolution analysis of the PCV2 ORF2gene of 47 strong positive samples showed that the predominant PCV2 strains in Hebei Province from 2015 to 2019 were PCV2b and PCV2d subtypes,but there were also a few PCV2e epidemics.2.The PCR detection method was used to amplify,clone,sequence and analyze the complete genes of two PCV2(QHD1PCV2-5,QHD2PCV2-182)and one PCV3(QHD3-PCV3)positive strains.The results show that QHD1-PCV2-5 belongs to PCV2e,QHD2-PCV2-182 belongs to PCV2d,and the homology with the reference strain is 93.3%-99.6%;it has high homology with the strains isolated after 2010,and the relationship is close,The accession numbers submitted to GenBank are MT711855 and MT711856.QHD-PCV3 belongs to the PCV3b subtype,and the GenBank accession number is MT71187.3.With reference to the complete sequences of PCV2 and PCV3 on GenBank,two pairs of specific circovirus detection primers were designed with Premier 5.0software,PCV2-F/R and PCV3-F/R,which were positive by gene amplification and other methods The tissue sample DNA was made into recombinant plasmids pMD-PCV2 and pMD-PCV3.The recombinant plasmid was used as a template to optimize the amount of primers and annealing temperature.The results show that the double nano-PCR method of recombinant plasmid pMD-PCV2 and pMD-PCV3 has the highest amplification efficiency when the temperature is 54.5?and the amount of primer is 1.0?L.Sensitivity test shows that the sensitivity of nano-PCR method is 100 times that of ordinary PCR.Among them,the minimum template detection amount of plasmid pMD-PCV2 and pMD-PCV3 is 1.2×10~3 copies/?L.Specificity test shows that there is no cross-reaction between viral nucleic acids.