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Development of a semiquantitative PCR assay using internal standard and colorimetric detection on microwell plate for pseudorabies virus.

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      Abstract

      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.

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      Affiliations
      [1 ] Centre National d'Etudes Vétérinaires et Alimentaires, Unité de Biologie Moléculaire, Ploufragan, France.
      Journal
      Mol. Cell. Probes
      Molecular and cellular probes
      Elsevier BV
      0890-8508
      0890-8508
      Dec 1997
      : 11
      : 6
      9500814 S0890-8508(97)90139-0 10.1006/mcpr.1997.0139

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