Multiplex polymerase chain reaction for the simultaneous detection and typing of polyomavirus JC, BK and SV40 DNA in clinical samples.

A novel multiplex nested PCR (nPCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK and SV40 in a single tube. In the first amplification step the same set of primers were used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV and SV40. The second round of multiplex nPCR was carried out using a set of primers designed to render products of different size for each related virus. The thermocycling parameters and concentration of each reaction component were optimised systematically to achieve optimal specificity and sensitivity for the nPCR assay. The sensitivity of the method ranged between one and 10 copies of polyomavirus genome. Cerebrospinal fluid (CSF) was examined from AIDS patients with clinical and neuroradiological evidence of progressive multifocal leukoencephalopathy (PML) and CSF from AIDS patients with other neurological alterations. Urine specimens from bone marrow transplant recipients affected by haemorrhagic cystitis were also tested. The results obtained suggest that the assay is a good tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and in other studies in order to define better the role of polyomaviruses in human disease.