We designed two pairs of primers and their corresponding TaqMan probes according to gH, gE gene of PRV. By optimizing the probe's concentration, Mg2+ concentration, primers concentration and sample DNA extraction, real-time fluorescent quantitative PCR (FQ-PCR) which can quickly identity field virus and vaccine virus of PRV was established. According to our results, the dynamic range of the FQ-PCR assay is between 10 x 10(1) copies/microL and 10 x l0(8) copies/microL, and the detection limit of FQ-PCR is 1.0 x 10(1) copies/microL, which is 100 fold higher than that of conventional PCR. We detected 60 doubtful tissue samples using the FQ-PCR assay, serum neutralization and conventional PCR. In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate, and can be used to detect field strains of PRV rapidly. The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2 h or less. Development of real-time quantitative PCR provides the basis for the early and rapid detection and analyzing quantitatively the infectious degree of PRV.
Animals ; Fluorescent Dyes ; Herpesvirus 1, Suid ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods ; Pseudorabies ; diagnosis ; prevention & control ; virology ; Pseudorabies Vaccines ; immunology ; isolation & purification ; Swine

Replication-incompetent adenoviruses expressing three major glycoproteins (gB, gC, and gD) of pseudorabies virus (PrV) were constructed and used to examine the ability of these glycoproteins to induce protective immunity against a lethal challenge. Among three constructs, recombinant adenovirus expressing gB (rAd-gB) was found to induce the most potent immunity biased to Th1-type, as determined by the IgG isotype ratio and the profile of the Th1/Th2 cytokine production. Conversely, the gC-expressing adenovirus (rAd-gC) revealed Th2-type immunity and the gD-expressing adenovirus (rAd-gD) induced lower levels of IFN-gamma and IL-4 production than other constructs, except IL-2 production. Mucosal delivery of rAd-gB induced mucosal IgA and serum IgG responses and biased toward Th2-type immune responses. However, these effects were not observed in response to systemic delivery of rAd-gB. In addition, rAd-gB appeared to induce effective protective immunity against a virulent viral infection, regardless of whether it was administered via the muscular or systemic route. These results suggest that administration of replication-incompetent adenoviruses can induce different types of immunity depending on the expressed antigen and that recombinant adenoviruses expressing gB induced the most potent Th1-biased humoral and cellular immunity and provided effective protection against PrV infection.
Adenoviridae/genetics/*immunology/metabolism ; Animals ; Antibody Formation ; Cell Line ; Cytokines/immunology ; Female ; Glycoproteins/biosynthesis/genetics/*immunology ; Herpesvirus 1, Suid/genetics/*immunology/physiology ; Immunity, Cellular ; Immunoglobulin G/immunology ; Mice ; Mice, Inbred C57BL ; Pseudorabies/*immunology/prevention & control ; Pseudorabies Vaccines/administration & dosage/*immunology ; Swine ; Th1 Cells/immunology ; Th2 Cells/immunology ; *Virus Replication