Chemiluminescent detection of amplified pseudorabies virus gp50 DNA with immobilized probes on microtiter wells.
A highly specific and sensitive PCR assay for the envelope glycoprotein gp50 has been developed for the detection of PRV-DNA sequences. Primer pairs from PRV gp50 gene were used with the enzyme uracil N-glycosylase and dUTP instead dTTP to prevent contamination due to PCR product carry-over. Biotinylated PCR products were captured in microtiter wells by specific oligonucleotide covalently linked to the polystyrene wells. After recognition of the biotinylated PCR product with a streptavidin phosphatase conjugate, a chemiluminescent detection was realised. Different factors influencing the binding, the hybridization and the detection efficiency have been tested.
Boutin
P
P
Centre National d'Etudes Vétérinaires et Alimentaires, Laboratoire de Biologie Moléculaire, Ploufragan, France.
Arnauld
C
C
Thiery
R
R
Costa
J M
JM
Vidaud
M
M
Jestin
A
A
D001483:Base Sequence; D001710:Biotin; D003124:Colorimetry; D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D011558:Herpesvirus 1, Suid / Q000235:genetics*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000235:genetics*
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral; D014759:Viral Envelope Proteins; C042966:pseudorabies virus glycoproteins; D001710:Biotin