Quantitation of polyomavirus DNA by a competitive nested polymerase chain reaction

A new method to quantitate small amounts of DNA in clinical specimens is described. The method, a nested competitive polymerase chain reaction (ncPCR), is able to quantitate between 10 and 10(6) copies per tube of polyomavirus DNA and shows good reproducibility when clinical samples are analysed. Throughout the whole procedure, an internal standard (IS) competes for the primers with the target DNA. The internal standard, a heterologous sequence containing the four primer recognition sites, was constructed using a modification of the 'MIMIC' approach that is useful for obtaining competitor sequences for any viral, bacterial or eukaryotic target. The ncPCR method for polyomavirus was applied to cerebrospinal fluids (CSF) from AIDS patients with progressive multifocal leukoencephalopathy (PML) and urine specimens from bone marrow transplant patients affected by haemorrhagic cystitis. The results obtained suggest that the ncPCR method is a sensitive and useful method for quantitating genomic load in clinical samples.