The Serological Investigation Of Swine Pseudorabie And Establish And Application Of FQ-PCR To Identify Wild Virus Or Vaccine Strain Of Porcine Pseudorabie Virus In Henan Province

In order to have a better understanding of the spread of PR in Henan province, this study, based on the serological statistics collected during the years from 2007 to 2014 in our lab, tried to group pigs in terms of time and place of birth and phase of growth and analyze the data from the perspectives of time, space and livestock.The comparative analysis showed that the g E antibody positive rate of PR was on the decrease year on year between 2007 and 2010. But since 2010, the figure has be rising steadily throughout the province. In the year 2014, 1373 pigs were tested for serum antibody statistics. After that, the data were classified in terms of region, i.e. east Henan, west Henan, north Henan, south Henan and central Henan. The results showed that north Henan suffered from the highest infection rate of PRV field virus which stood at 66.67%. Following are central and south Henan with the infection rate being 59.51% and 53.9% respectively. In the same year, serological statistics with clear backgrounds were collected from 766 pigs. The data were then grouped based on the different feeding phases. The results showed that the positive rates for sows and backup sows were respectively 64.21 and 50%. What followed were fattening pigs with the field virus positive rate being 49.01%.Although vaccination has been an effective way to prevent and control the spread of PR, the latter’s occurrence has become increasingly high in the recent years. This has posed an important question as to how to distinguish field viruses from vaccine strains. The current study, in accordance with the PRV-gD/gE genetic serial in Gen Bank, selected 2 specific primers and 2 MGB probes from the highly conservative serial design. The matrix method was adopted to screen for the best primer and probe concentration and to optimize the best annealing temperature. Before that, tests were conducted for its sensitivity, specificity, repetitiveness and stability. In the meantime, clinical tests were given to the suspected samples infected by PRV and the results were then compared by means of routine PCR.The results showed that after being optimized the best concentration combinations PRV-gE/p1 and PRV-g D/p1/p2 were both 0.7μmol/L and that the concentration of PRV-gE/p2 was 0.8μmol/L; Probe-PRV-gE was 0.4μmol/L and ProbePRV-gD was 1.4μmol/L; the optimal annealing temperature was 60℃. The detection limit for pGEM-T/PRV-gE was 1×101copies/μL and it shared the same sensitivity as PCR. The specificity tests done on 9 viruses and 3 vaccines has showed that this method features good specificity and can be used for the differentiation between field viruses and vaccine strains. Mixing up the same amount of plasmids of p GEM-T/PRVgE and pGEM-T/PRV-g D, To repeat respectively 3 times of 1×107、1×105、1×102copies/μL,we successfully gave 3 tests to 1×107、1×105、1×102copies/μL respectively, proving the good repetitiveness of the method. 55 suspected positive samples were tested by means of double FQ-PCR and routine PCR. The former recognized 30 positive samples, among which 19 had double positive genes and 11 gD positive. The routine PCR test identified 25 positive samples with 15 having double positive genes and 10 having gD positive. It’s fair to say that the double FQPCR test is more sensitive and effective in identifying positive samples and can be used to distinguish isolated pseudorabies viruses from vaccine strains. If the amplification result of gD is positive and that of gE is negative,we can be sure that PR vaccine is infected; if, on the other hand,both the amplification curves of gD and g E are positive, then PR field virus is infected.This method is of great significance in that it can not only help to diagnose earlystage PRV infection but also help to differentiate field viruses from vaccine strains.