Porcine Circovirus Type2Fluorescence Quantitative Pcr Detection Method To Establish And Nurture The Preliminary Study Of The Process

There are two kinds of genotypes of porcine circovirus, PCV1and PCV2, PCV1is not pathogenic, it can infect PK-15cells chronically and is considered to be a cell contaminant in the early times. PCV2is a major pathogen of causing postweaning multisystemic wasting syndrome(PMWS).It can lead to porcine reproductive failure, porcine dermatitis and nephropathy syndrome and porcine respiratory disease syndrome and other diseases, these diseases are often referred to as PCV2-related diseases. Since the discovery of PMWS, the diseases brought by PCV2has brought huge losses to the pig industry.This study was aimed to establish two detection methods of fluorescence quantitative PCR and indirect immunofluorescence. The main contents and results were as follows:(1)Construction of Recombinant plasmid pMD-ORF2:A pair of primers were designed according to ORF2gene sequences of porcine circovirus type2(PCV2) in GenBank, in order to facilitate restriction enzyme digestion and connect to the expression vector. BamHl restriction sites were introduced in upstream of the primer and Hind III restriction sites in downstream. ORF2gene of PCV2DNA was amplified by PCR (Total length is702bp). Then this gene fragment was connected to into pMD20-T vector, recombinant plasmid the pMD-ORF2was screened after PCR and restriction enzyme digestion.(2)Establishment of Fluorescence quantitative PCR(FQ-PCR) for PCV2detection: A pair of specific primers and a TaqMan probe were designed based on the conservative fragments of PCV2ORF2gene sequence. The FQ-PCR assay was carried out by quantitative concentration of serial10fold dilutions of pMD-ORF2DNA by optimizing, circulation parameters. The results showed that the sensitivity of the method is up to1.0× 101copies/uL and100times higher than traditional PCR, and stated that the method had good accuracy and sensitivity.PK-15cells are often used as a host cell where PCV2proliferates in vitro, PCV2can exist in vivo as a long-term high titer, but its proliferation titer is quite low and unstable in vitro.This experiment is to improve PCV2titer in vitro,preliminary studying from the four aspects:the time of harvest virus,addition of D-glucosamine in vitro,stability training and different culture systems.It is found that PCV2copy number in72h was significantly higher than the virus copy number of24h and48h after virus infection;the PK-15cell with PCV2in vitro without D-glucosamine; because its proliferation titer is quite low and unstable in vitro,so this experiment chose infected continuous subculture in the cell until the fourth generation,virus copy number begin to gradually stable and constantly increase,while in the experiment it is also found that PCV2is a strong host dependent;in a small square bottles,roller bottles and microcarriers three culture systems,roller bottles and microcarrier of PCV2TCID50is cultured up to10-7/mL.