Development of a semiquantitative PCR assay using internal standard and colorimetric detection on microwell plate for pseudorabies virus.
We have developed a semiquantitative PCR assay on microtitre plates for quantitation of pseudorabies virus (PRV). The test is based on co-amplification with an internal control (IC) of the target viral DNA, followed by hybridization of the biotin-amplified products on a capture probe covalently immobilized to a Covalink-NH MicroWells plate and then visualization with colorimetric enzymatic reactions. PCR was performed in the presence of uracil-N-glycolsylase (UNG) with dUTP instead of dTTP to prevent false positive results due to carry-over contamination. Our colorimetric test had a 3.5 log dynamic range with a detection level of 30 DNA copies per PCR reaction. A standard curve for quantitation of pseudorabies virus was established from co-amplification of 10 to 10(5) PRV molecules with 1000 IC molecules. Ratios of viral optical density/IC optical density were plotted against the number of PRV DNA target molecules in the PCR amplification. Integration of 96-well formats and automation using robots at different steps of the test ensured a good repeatability. Calibration of the quantitative test using samples from experimentally-infected pigs is in progress.
Denis
M
M
Centre National d'Etudes Vétérinaires et Alimentaires, Unité de Biologie Moléculaire, Ploufragan, France.
Soumet
C
C
Legeay
O
O
Arnauld
C
C
Bounaix
S
S
Thiéry
R
R
Jestin
A
A
D001331:Automation; D001483:Base Sequence; D003124:Colorimetry / Q000379:methods*; D011558:Herpesvirus 1, Suid / Q000235:genetics*; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D014773:Virology / Q000295:instrumentation / Q000379:methods