Development of Real-Time Polymerase Chain Reaction Assays for Rapid Detection and Differentiation of Wild-Type Pseudorabies and Gene-Deleted Vaccine Viruses

Abstract

The successful eradication of pseudorabies in U.S. domestic swine was accomplished through the use of glycoprotein E (gE) deleted modified live virus vaccines and an accompanying gE differential enzyme-linked immunosorbent assay (ELISA). Yet, pseudorabies virus (PRV) was established in feral swine in the United States, becoming a potential reservoir of PRV for infection of domestic swine and other native wildlife. A critical need for the current PRV surveillance program in the United States is the rapid detection of PRV infection. For this reason, a set of 2 real-time polymerase chain reaction (PCR) assays by using TaqMan chemistry was developed and evaluated for their capability in the detection and differentiation of field and vaccine strains of PRV. PCR primers and probes were designed for gB and gE genes of PRV, respectively. The newly developed PRV-specific real-time PCR assays could detect all wild-type PRV isolates from diagnostic submissions and differentiate them from vaccine strains. The analytical sensitivity of the assays was approximately 0.1 plaque-forming units per reaction. The assays were highly specific for PRV, because no positive results were obtained from testing other common swine viral pathogens and other animal herpesviruses. The results of testing samples from domestic and feral swine and from bovine showed that the real-time PCR assays are more sensitive than gel-based PCR. These results demonstrated the potential application of the developed real-time PCR assays as a differential test for rapid and specific detection of PRV in domestic and feral swine, as well as nonporcine species that can be infected with PRV and serve as carriers.

Different Shedding Patterns Measured by Virus Isolation and Real-Time PCR in Pigs Challenged with Aujeszky’s Disease Virus

“…There was agreement between the results of the qPCRs targeting gB or gE, as demonstrated by the individual measurements on 5 DPC and alike the data published on the used protocol ( Figure 2) [9]. …”

“…The same samples were further processed by qPCR capable of discriminating wild-type and gE-deleted vaccine viruses, having a lower sensitivity for detection of the gE gene [9]. Briefly, nucleic acid was extracted from the swabs by QIAxtractor nucleic acid purification device using DX Reagent Pack (Qiagen) according to the manufacturer's protocol.…”

“…Briefly, nucleic acid was extracted from the swabs by QIAxtractor nucleic acid purification device using DX Reagent Pack (Qiagen) according to the manufacturer's protocol. Quantity of ADV was determined with qPCR using the gB-specific primers and probe (labelled with FAM) as described by Ma et al (2008). Results of the qPCR assay were analysed as Cq values.…”

Different methods of real-time PCR for detection of pseudorabies virus

“…A nested-PCR (FONSECA JR et al, 2010a) tested using the same samples to test analytical sensitivity had the same results for gD-plexor, was one log more sensible than gB-SYBR and one log less sensible than gB-Taq. Two hydrolysis probe qPCR for gE and gB described by MA et al (2008) presented similar results, with gE less sensible than gB, but no PCR was able to amplify samples stored for more than 10 years as done in this research These PCRs, as well as others (YOON et al, Table 2 -Statistics generated from the Cts of qPCR-gB-Taq using samples at a concentration of 200 ng/uL. were not subjected to all steps in a validation such as described here (repeatability and reproducibility, interlaboratorial tests).…”

La biotecnología en sanidad animal

“…Ejemplos de estos inmunoensayos son el ELISA de detección de Anaplasma (Trueblood, McGuire y Palmer, 1991), del virus de la Diarrea Viral Bovina (Mignon et al, 1991) y del virus de la Peste Bovina y del virus de la Peste de los Pequeños Rumiantes (Libeau et al 1994) para detección del patógeno. Respecto a los ELISAs de detección de anticuerpos, han tenido gran importancia los ensayos DIVA utilizados en la erradicación del virus de la Pseudorabia en porcino (Ma et al, 2008) o en la erradicación de la Influenza Aviar de baja patogenicidad en aves domésticas (Marangon et al, 2003) entre otros.…”