Quantitation of polyomavirus DNA by a competitive nested polymerase chain reaction

A new method to quantitate small amounts of DNA in clinical specimens is de scribed. The method, a nested competitive polymerase chain reaction (ncPCR) , is able to quantitate between 10 and 10(6) copies per tube of polyomaviru s DNA and shows good reproducibility when clinical samples are analysed. Th roughout the whole procedure, an internal standard (IS) competes for the pr imers with the target DNA. The internal standard, a heterologous sequence c ontaining the four primer recognition sites, was constructed using a modifi cation of the 'MIMIC' approach that is useful for obtaining competitor sequ ences for any viral, bacterial or eukaryotic target. The ncPCR method for p olyomavirus was applied to cerebrospinal fluids (CSF) from AIDS patients wi th progressive multifocal leukoencephalopathy (PML) and urine specimens fro m bone marrow transplant patients affected by haemorrhagic cystitis. The re sults obtained suggest that the ncPCR method is a sensitive and useful meth od for quantitating genomic load in clinical samples. (C) 2000 Elsevier Sci ence B.V. All rights reserved.