PCR reaction was carried out in 20 microliter capillaries containing master mix (6mM this site MgCl2, 0.1mg/ml BSA (Sigma, St. Louis, USA)) and 10 picoM of each primer (Metabion, Martinsried, Germany), 3 picoM of each hybridization probe (5��-GGCTGCTGCTGCCACAGGATTTTC-FL (probe 1) and 5��LC Red640-GTAGCTGAAATTGCTGCTGGAGAG-ph (probe 2), 0.25mM dNTP’s (Roche)), and 1U Platinum Taq Polymerase (Invitrogen, Karlsruhe, Germany). Target DNA was added in a volume Inhibitors,Modulators,Libraries of 5 microliters. Two negative and 4 positive controls in different concentrations were included into each run performed on a LightCycler 1.0 instrument (Roche, Mannheim, Germany) under the following conditions: initial denaturation and activation of hot-start enzyme at 95��C for 30s, followed by 45 cycles of denaturation at 95��C for 0s, annealing of primers at 58��C for 10s, and primer extension at 72��C for 20s.
Fluorescence was monitored by Inhibitors,Modulators,Libraries single acquisition during the annealing phase of each PCR cycle and channel setting F2/F1 (640nm/530nm). Differentiation of BK virus amplicons from JC virus depends on the binding of the probe 2, which perfectly matches JC virus but differs at the two underlined positions from BK virus. Thus binding to BK virus results in a shifted melting curve (61.5��C for BK versus 63.8��C for JC). For detection of the viral large T-antigen, a 128bp long fragment was amplified using modified primers from [18] that match BKV genotypes 1 to 4, forward primer 5��-CAGGCAAGGGTTCTATTACTAAAT-3��, reverse primer 5��-GCAACAGCAGATTCYCAACA-3��, probe 5��-6FAM-AAACTGGTGTAGATCAGARGGAAAGTCTTTAGGGTCTT-BBQ.
PCR conditions were identical to the one described above except TaqMan probe that was used at 4 pico molar, Inhibitors,Modulators,Libraries primer annealing was 56��C for 10s, and fluorescence acquisition was at the end of each Inhibitors,Modulators,Libraries primer extension phase at 530nm. 4. Discussion We described a young patient with precursor T-ALL who had received an unrelated matched donor PBSCT and one year later presented with increase creatinine levels and bilateral hydronephrosis. Nephrological and urological examination showed an epithelial proliferation of bladder on both ostii being responsible for bilateral hydronephrosis. Further pathological and molecular analysis revealed widespread endothelial infection by a BK virus. We speculate that the endothelial cell injury led to capillary injury, oedema, and tumorous proliferation on both ostii.
Other organs were not involved. This case demonstrated Inhibitors,Modulators,Libraries a very late (one year after MUD-HSCT) proliferative endothelial infection by a virus related to severe viral load and hemorrhagic cystitis 4 weeks after unrelated PBSCT. Although the tumour mass could not be unambiguously attributed to BKV since the staining of large T antigen was negative in immunohistochemical analysis, large T-cell antigen and BK virus were positive by PCR, thus supporting AV-951 the view that BK virus may indeed play a role in the development of this tumour.