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 wer e 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 relat ed virus. The thermocycling parameters and concentration of each reaction c omponent were optimised systematically to achieve optimal specificity and s ensitivity for the nPCR assay. The sensitivity of the method ranged between one and 10 copies of polyomavirus genome. Cerebrospinal fluid (CSF) was ex amined from AIDS patients with clinical and neuroradiological evidence of p rogressive multifocal leukoencephalopathy (PML) and CSF from AIDS patients with other neurological alterations. Urine specimens from bone marrow trans plant recipients affected by haemorrhagic cystitis were also tested. The re sults obtained suggest that the assay is a good cool for supporting the dia gnosis of polyomavirus infection and could be used for epidemiological purp oses and in other studies in order to define better the role of polyomaviru ses in human disease. (C) 1999 Elsevier Science B.V. All rights reserved.