Establishment And Preliminary Application Of Multiplex PCR Assay For Detection Porcine Adenovirus

Porcine adenovirus was first isolated in 1964 in England, and thereafter, it was proved that the porcine adenovirus was widespread in the pigs by virus isolation and serological survey. However the harm as a natural adenovirus infection pathogens,its economic losses as well as the adenovirus so far has not fully been understood. Current diagnostic tools used to test porcine adenoviruses are porcine viruses isolation, direct immunofluorescence, gel immunodiffusion test, hemagglutination, hemagglutination inhibition and virus neutralization test, molecular biology methods. Virus isolation, and direct immunofluorescence test were relatively complicated, and cost more money and time, and are hard to conduct quickly and easily a large-scale porcine adenovirus research. At present, scholars abroad and domestic detect this adenovirus using porcine molecular biology methods, but this has not become routine PCR detection methods. The aim of this study is to make investigation on the pathogenic prevalence and to establish a rapid method of detection of porcine adenovirus.Establishment of a multiplex PCR assy for detection porcine adenovirus:First finding the four serotypes gene sequence of the PADV on the NCBI GenBank, and conservative analyszed the gene sequence homology by the DNAstar software, and got the gene sequence homology. Designing Primer of four sertypes based on conserved sequences with software Primer5.0. Finally, the PCR primer concentration, annealing temperature optimization and sensitivity and specificity analysis. As a result,all of the genes, designing primers of PADV3 and PADV5 in conservative parts of the hexon, designing primers of PADV1 gene near ORF1-5 area, and that of PADV4 in E1B region. We find the multiplex PCR is the best in Each primer concentration in 0.6umol, annealing at 55 ℃, and found that the multiplex PCR method has good specificity through virus Detection of the PEDAV、PTEGV、PRRSV、 PCV、PIV and PASTV.The preliminary application of multiplex PCR for porcine adenovirus:based on the tests of 318 samples tested porcine anal swabs,80 parts of pig lungs, nasal swabs 173 pigs, 180 porcine serums. The result show that the positive rate is 20.75% anal swabs, the positive rate of nasal swabs is 10.4%, the positive rate of porcine is serum 1.66%, the positive rate of lung positive was 7.5%. The positive samples were detected with gene sequencing, and the gene on NCBI are comparative analyzed has good specificity and reproducibility and can be used for detection of porcine adenovirus which provides important technical support for the investigation and diagnosis of adenovirus infection. From the test results found in diseased pigs infection than health pigs, pig no significant difference in the prevalence of each season, Breeding environment difference farms infection than good breeding environment.