Free Access
Vet. Res.
Volume 31, Number 1, January-February 2000
Page(s) 119 - 120
How to cite this article Vet. Res. (2000) 119-120
Vet. Res. 31 (2000) 119-120

Development and evaluation of TaqMan-based PCR assays to quantitate PRV latency

N. Alemana, A. Vilnisb, H. Hillc, B. Thackerd and R. Maesb, e

a  Dept. Anatomia Pathologica, Faculty of Veterinary Medicine, Lugo, Spain
b  Dept. of Microbiology, Rm. A45 Veterinary Medical Center, Michigan State University, E. Lansing, MI 48824, USA
c  Murphy Family Farms, Rose Hill, NC 28458, USA
d  Dept. of Vet. Clinical Sciences, Iowa State University, Ames, IA, USA
e  Animal Health Diagnostic Laboratory, Michigan State University, E. Lansing, MI 48824, USA

Abstract - Aujeszky's disease virus (ADV), also referred to as pseudorabies virus (PRV), is a herpesvirus classified under the subfamily of Alphaherpesvirinae. As a result of successful eradication efforts, the incidence of ADV infections has been highly reduced, but in some areas this virus still causes significant economic losses due to baby pig mortality, respiratory disease in feeder pigs and reproductive losses in breeding age swine. Latency is an integral part of the ecology of herpesviruses. Reactivation of latent ADV DNA results in renewed replication and potential transmission to susceptible pigs. Our laboratory has been involved in defining the influence of vaccination with commercially available ADV vaccines on the relative level of latency following experimental challenge with virulent ADV. The development of quantitative PCR (QPCR) methods was an essential part of these studies. In our previously reported experiments we used chemiluminescence-based PCR assays, specific for the ADV gG and gE genes. With this QPCR system we determined the influence of vaccine genotype and the route of administration on the protection of previously vaccinated pigs against field virus latency. Five genotypically distinct modified-live ADV vaccines, administered IM or IN, were used in these studies. Field virus group mean latency load in a group of unvaccinated pigs exposed to virulent ADV was $118000 \pm 41200$ copies per microgram of total ganglionic DNA. Vaccination with all but one vaccine prior to challenge significantly reduced field virus latency loads. The extent of the reduction was dependent both upon the vaccine strain and the route of administration. A recently introduced TaqMan $^{\ooalign{\hfil\hbox{\textsc{r}}\hfil\crcr\mathhexbox20D}}$ QPCR utilizes a fluorogenic probe, designed to measure PCR product formation in real time. The fluorogenic TaqMan $^{\ooalign{\hfil\hbox{\textsc{r}}\hfil\crcr\mathhexbox20D}}$ probe consists of an oligonucleotide labeled at its 5' end with a reporter molecule and at its 3' end with a quencher. During the PCR reaction the TaqMan $^{\ooalign{\hfil\hbox{\textsc{r}}\hfil\crcr\mathhexbox20D}}$ probe anneals to its target and is cleaved by the 5' exonuclease activity of the Taq polymerase. This results in the release of the reporter and the generation of a sequence-specific fluorescent signal. Unlike other QPCR systems, this real-time detection system quantitates at early cycles, when the fidelity of the PCR reaction is highest. Another advantage of this approach is that it provides a dynamic assay range of at least five orders. Three groups of experimental pigs were used in this study. The first group consisted of 10 feeder pigs, vaccinated IM with a commercial TK -, gE -, gG - PRV vaccine. Thirty days post-vaccination the experimental pigs in this group were exposed orally to 105 TCID 50 of a virulent strain of PRV. The second group consisted of 6 feeder pigs, which were vaccinated but not challenged. A third group of 3 pigs was not vaccinated or challenged and kept as environmental controls. Left and right trigeminal ganglia were collected from all pigs on day 60 of the experiment. Each trigeminal ganglion was cut into 4 pieces. Total DNA from each piece was extracted using a commercial kit and quantitated by fluorometry. Primers and probes for the TaqMan $^{\ooalign{\hfil\hbox{\textsc{r}}\hfil\crcr\mathhexbox20D}}$ QPCR were designed with the Perkin Elmer Primer Express $^{\ooalign{\hfil\hbox{\textsc{r}}\hfil\crcr\mathhexbox20D}}$ program. Since it is not target specific, and also more economical, SYBR $^{\ooalign{\hfil\hbox{\textsc{r}}\hfil\crcr\mathhexbox20D}}$ green-based assays were used to define Mg ++ optimum, primer concentrations and reaction conditions. Optimized reaction mixes for both the gE and gD-specific SYBR $^{\ooalign{\hfil\hbox{\textsc{r}}\hfil\crcr\mathhexbox20D}}$ green assays included $1 \times$ master mix, 300 nM of each primer, 150 nM of the probe and 100 ng of DNA template. The cycling profile for both assays consisted of $50\,^\circ$C for 2 min, $95\,^\circ$C for 10 min, followed by 45 cycles composed of a denaturation at $95\,^\circ$C for 15 s and annealing/ elongation at $60\,^\circ$C for 60 s. Using the current assay conditions we can reliably detect 10-100 copies of purified ADV DNA, both with the gE- and gD-specific primers. The specificity of the amplicons was verified by sequencing. Probe-based assays maximize assay specificity. gE- and gD-specific, 6-FAM and TAMRA labeled probes were synthesized for this purpose. The probe concentration optima were 150 nM for both probes and the detection limits of the TaqMan $^{\ooalign{\hfil\hbox{\textsc{r}}\hfil\crcr\mathhexbox20D}}$ probe assays were 10-100 copies of ADV DNA. Using standard PCR on DNA aliquots from all of the 80 ganglion pieces, we confirmed our previous observation that the latency load varies between pigs and between individual ganglion pieces from each pig. Using the gE-specific assay, we determined that the group mean latency load of 10 vaccinated and challenged pigs was $6080 \pm 3325$ copies of virulent ADV DNA per microgram of total ganglionic DNA. The latency load in the group that was vaccinated but not challenged, as determined by the gD-specific assay, averaged approximately 100 copies per microgram of ganglionic DNA. This was at the very limit of assay sensitivity. Latent ADV DNA was not detected in ganglionic DNA extracts of any of the control pigs. In conclusion, we have developed sensitive and convenient TaqMan $^{\ooalign{\hfil\hbox{\textsc{r}}\hfil\crcr\mathhexbox20D}}$-based PCR assays to quantitate latent ADV DNA. The gE-specific assay was used to quantitate the virulent ADV latency load in the trigeminal ganglia of 10 pigs, which were first vaccinated with a TK -, gE -, gG - commercial PRV vaccine and subsequently exposed to a high dose of virulent PRV. The low group mean virulent ADV latency load detected after challenge of pigs vaccinated with this particular vaccine was similar to the one in our previous study. The gD-specific assay, used in conjunction with the gE-specific assay, would allow quantitation of latent vaccine virus DNA load in the samples, provided it is above the sensitivity threshold of the TaqMan $^{\ooalign{\hfil\hbox{\textsc{r}}\hfil\crcr\mathhexbox20D}}$ probe assay.

Corresponding author: R. Maes Tel.: (1) (517) 353 22 96; fax: (1) (517) 353 44 26;

© INRA, EDP Sciences 2000