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 fa r quantitation of pseudorabies virus (PRV). The test is based on co-am plification with an internal control (IC) of the target viral DNA, fol lowed by hybridization of the biotin-amplified products on a capture p robe covalently immobilized to a Covalink(R)-NH MicroWells(R) plate an d then visualization with colorimetric enzymatic reactions. PCR was pe rformed in the presence of uracil-N-glycolsylase (UNG) with dUTP inste ad of dTTP to prevent false positive results due to carry-over contami nation. Our calorimetric test had a 3.5 log dynamic range with a detec tion level of 30 DNA copies per PCR reaction. A standard curve for qua ntitation of pseudorabies virus was established from co-amplification of 10 to 10(5) PRV molecules with 1000 IC molecules. Ratios of viral o ptical density/IC optical density were plotted against the number of P RV DNA target molecules in the PCR amplification. Integration of 96-we ll formats and automation using robots at different steps of the test ensured a good repeatability. Calibration of the quantitative test usi ng samples from experimentally-infected pigs is in progress. (C) 1997 Academic Press Limited.