The Gene Clone Pcmv Dpol Analysis Based Pcr Method For Detection Of This Gene To Establish Initial Application

According to the published sequence of DNA Polymerase (DPOL) Gene of Porcine Cytomegalovirus (PCMV) (Genbank accession number AF268040), a pairs of specific PCR primers were designed to amplify the gene sequence, and the target gene was linked into pMD19-T vector. The sequencing analysis revealed the whole PCMV DPOL gene was an open reading frame consisting of 3024 bp in length and had a length of 1008 amino acids residues. The nucleotide sequence of PCMV DPOL gene shared an approximately 98% identity with the referred sequence, and phylogenetic tree analysis showed that PCMV had a closely relationship with III IV6 and HHV7. Lots of physical and chemical properties of the deduced protein from the DPOL gene were performed by several bioinformatics analysis software, and its advanced structure was predicted.Two pairs of primers were designed according to DPOL gene of PCMV to develop a nest PCR assay for detection of PCMV, the longth of the product is 236bp. Test the specificity, sensitivity and repeatability of the PCR assay, the result indicate that the necessary dose of templates of the assay is 1.lpg and this method is sensitive specific and repeatable.The positive rate of the 36 clinical samples which are PRRSV positive, detected according to the method, was 63.89%.Four pairs of primers were designed according to DPOL gene of PCMV, gE gene of PRV, VP2 gene of PPV and PCV2 ORF2 to develop a multiplex PCR(m-PCR) assay for detection of PCMV, PRV, PPV and PCV2, the longth of the products are 250bp,380bp,500bp and 660bp. Optimized the multiplex PCR amplification reaction condition through change the reaction buffer, the different composition of primers dilution, the different Taq DNAase dilution and the the different composition of dNTP and Mg2+dilution. At the same time, a comparative study was performed with two different template DNA extract ways of PCMV, it found that DNA extracted by Kit is the better one, by Phol-ChI also can ensure the right result. The necessary dose of templates of the assay is 10-1pg and this method is sensitive specific and repeatable. Compare the m-PCR to GB/T 18641-2002 (PRV), SN/T 1874-2007(PPV) and GB/T 21674-2008(PCV2) though detected the same 50 samples, the result indicated that the sensitivity of the m-PCR assay is 100%, the specificity is 97.5% for PRV,100% for PPV and 97.4%for PCV2, the coincidence rate is 98%for PRV,100% for PPV and 98% for PCV2. In this study,123 samples which were reproductive disorder were detected by this m-PCR method, it showed that four kinds of viruses is popular, especially PCMV and PCV2, the rate of positive sample were 34.1%and 32.5%. In case of two kinds of viruses combined infection, PCMV and PCV2 mixed infection is serious, the rate of positive sample is 13.8%. The highest rate of three kinds of viruses combined infection is PRV, PCMV and PCV2, is 6.5%. There is only one sample is infected the four kinds of viruses the same time.