Isolation, Identification And Development Of SYBR Green â…  Real-Time PCR Assay And Clinical Detection Of Porcine Parvovirus

Porcine parvovirus (PPV) is one of the most important pathogens responsible for reproductive disorders including stillbirth,mummification,embryonic death and infertility. After infecting primiparous pregnant sows and then invaded embryonic or fetal placenta,causing sow abortion,fetal death,fetal malformation and the mummy,resulting in infertility or repeated estrus for sows,piglets can also cause dermatitis and diarrhea. PPV widespread and prevalent in farms at home and abroad,seriously affecting and constraining the development of the pig farming industry. PPV are often associated with porcine pseudorabies virus,porcine circovirus typeⅡ,porcine Japanese encephalitis virus,porcine reproductive and respiratory syndrome virus co-infection and thus increase the hazard. In recent years,PPV infection and the harm caused by the upward trend to the pig farming industry and caused great economic losses. Therefore,to enhance the PPV of the basic research it is very important. To this end,we carried out some researches:1. To deal with collecting porcine parvovirus infection in patients expected and extract DNA. According to the reported PPV NADL-2 genome sequence,were designed and synthesized a pair of oligonucleotide primers,by optimizing the PCR reaction conditions,we successfully amplified 195bp fragment from the suspected disease,recovered PCR product sequencing and digestion with EcoRⅠ,the result is the same to expected. The PRV DNA,PPV DNAand PK-15 cells normal genomic DNA were negative for the PCR amplification confirmed that the specificity of the amplified fragment. To deal with the supernatant and then inoculate PK-15cells,after cultivating and proliferating more than generation of times,we uccessfully isolated two porcine parvovirus strains,named HN-2,HN-3 separaetedly,and studied their biological characteristics of the virus. The results show thatHN-2 strain, HN-3 strain. on ether,chloroform,trypsin,pH3.0 acid,and 56℃heat treatment are not sensitive. HN-2 strain and HN-3 strain can result in stability cytopathic effect in PK-15 cells, the electron microscopy results show that the virus particles are round and 26nm in diameter,confirmed that the two strains isolated from porcine disease are porcine parvovirus indeed.The field porcine parvovirus for the new vaccine research and development provide a reference strains.2. we extracted the virus DNA frome HN-2 strain and HN-3 strain that the PK-15 cell proliferation,designed two pairs of synthetic oligonucleotide primers according to the reported PPV NADL-2 genome sequence in GenBank and amplified porcine parvovirus non-structural protein NS1 gene and structural protein gene VP2,finally,successfully amplified fragments of the NS1 gene 1620bp and 1438bp fragments of VP2 gene,recovering and sequencing of PCR products,using the software of the DNAstar to analysis the clonning sequences and compare with the published in GenBank. PPV corresponding gene sequence homology close to 99.5% , respectively,99.7%,confirmed that the virus isolated strains from porcine are porcine parvovirus once again,this research provides a theoretical basis and foundation for PPV molecular biology for in-depth study.3. We plucked porcine heart,lung,kidney,spleen,liver,lymph nodes,uterus and other organs which infected by PPVand DNA were extracted from aborve organs and tissues.Using the established porcine parvovirus SYBR GreenⅠreal-time fluorescent PCR detection assay to detect the relative content of PPV DNA in tissues and comparing and analysising the PPV DNA content in the different organs of the same porcine or the PPV DNA content in the same organs of the different pigs,The results showed that heart,liver,spleen,kidney,lung,lymph nodes,uterine pregnancy sows all contain porcine parvovirus,including kidney,lung,lymph nodes,uterus of pregnant sows is relatively high for PPV content,the content of the liver relatively low,thus we believe that the PPV of the kidney,lung,lymph nodes,the pro-uterine pregnancy sows have the strongest tropism,but for the liver tropism of the pro-less. This assay also applificate SYBR GreenⅠreal-time fluorescent PCR to study a total of PPV and PCV2 infections,confirmed the existence of PCV2 infection can promote the PPV to prolifer and replicate,PCV2 can promote and enhance the PPV of the pro-infected tissue tropism. The sensitivity of the PPV SYBR GreenⅠreal-time fluorescent PCR detection method is 79copies/μL,be used to study the content and distributione in the main organs for the field PPV,Supply the heoretical and experimental basis to research the PPV molecular pathogenesis and pathogenic mechanisms,but also for the early diagnosis and clinical testing of PPV provides a fast,sensitive,specific, qualitative,quantitative,low-pollution rate detection assay.