A fluorescence-based quantitative PCR method for investigation of pseudorabies virus latency.
A quantitative PCR method was developed in order to quantitate the number of copies of Pseudorabies virus (PRV) genome present in tissues from infected pigs. The method is based on the use of an internal standard that differs from the target DNA by a deletion of ten base pairs, and that is co-amplified with the target DNA. The resulting PCR products are labelled with a fluorescent primer and are then separated and detected by means of an automated sequencer. The assay was found to be specific and sensitive, allowing the detection of five copies of viral DNA among 10(6) host cells. The method was used successfully to quantitate the number of PRV DNA copies in trigeminal ganglia samples from infected pigs during the acute and the latent stages of the infection. Between 12 and 3.10(5) copies of viral genome per 10(6) neuronal cells were detected in these tissues which is consistent with data published previously.
Thiery
R
R
Centre National d'Etudes Vétérinaires et Alimentaires, Laboratoire de Biologie Moléculaire, Ploufragan, France. rthiery@citi2.fr
Pannetier
C
C
Rziha
H J
HJ
Jestin
A
A
D000818:Animals; D004279:DNA, Viral / Q000032:analysis*; D005453:Fluorescence; D018628:Gene Dosage; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011557:Pseudorabies / Q000821:virology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine / Q000821:virology; D012668:Trigeminal Ganglion / Q000473:pathology / Q000821:virology; D014759:Viral Envelope Proteins / Q000235:genetics*; D017735:Virus Latency
D004279:DNA, Viral; D014759:Viral Envelope Proteins; C042966:pseudorabies virus glycoproteins