Rapid quantification and differentiation of human polyomavirus DNA in undiluted urine from patients after bone marrow transplantation

A combined PCR assay was developed for the detection and typing of human po lyomavirus (huPoV) in clinical samples, consisting of (i) a qualitative sem inested PCR assay (snPCR) to discriminate between huPoV EEL and JC and (ii) a high-throughput, quantitative TaqMan PCR assay (TM-PCR) for the general detection of huPoV. The TM-PCR detects huPoV DNA in a linear range from 10( 7) to 10(1) copies per assay. In reproducibility runs, the inter- and intra -assay variabilities were less than or equal to 60 and less than or equal t o 50%, respectively. The snPCR assay uses a set of four primers for the sam e region of the BK and JC viral genomes, In the first round of amplificatio n, two general primers were used; in the second round, one of these general primers and two additional, BK- or JC-specific primers were used simultane ously to produce amplicons of different sizes specific for BK virus (246 bp ) and JC virus (199 bp), respectively. We tested different urine dilutions in order to determine the inhibitory effects of urine on PCR amplification. Furthermore, we compared the use of native urine with DNA purified by diff erent preparation procedures. Our results show, that a 1:10 dilution of the urine led to complete reduction of the amplification inhibition found with 6% of undiluted urine samples. In a clinical study including 600 urine spe cimens, our assay turned out to be fast, cheap, and reliable in both qualit ative and quantitative aspects.