For the detection of TSV DNA, three real-time quantitative PCRs were developed with primers and Taqman probes located in the NCCR, and the VP1 and Large T ORFs, respectively (Table S2 ). Primers and probes were chosen with the help of Beacon Designer software (Premier Biosoft). The VP1 3′ primer had a 84% match with BKV, but none of the chosen TSV probes had similarities with any of the other known polyomaviruses.
The 50 µl PCR reactions consisted of 1× GeneAmp PCR buffer (15 mM Tris-HCl [pH 8,0], 50 mM KCl, 3,6 mM MgCl, 0,3 mM of each dNTP, 15 pmol of each primer, 7,5 pmol probe and 2 U of AmpliTaq Gold polymerase (Applied Biosystems). Real-time PCR was performed in the iCycler (Biorad) and cycle conditions are 9′ at 95°C, followed by 50 cycles of amplification (94°C for 1 min. and 65°C for 1 min.). TSV copy number was calculated against a plasmid titration series of pUC19-TSV included in each PCR assay that contains the full length TSV genome cloned in XbaI. Cell number was calculated with a PCR specific for the beta-actin gene, which was run in parallel on a dilution series of human genomic DNA (Promega). The sensitivity of each TSV PCR was found to be between 1–10 TSV genome copies.
The cerebrospinal fluids, blood plasmas and respiratory samples with proven JCV-, BKV-, KIV- or WUV-positivity used for validation of the developed TSV-specific PCRs were selected from clinical samples routinely send for viral diagnosis to the LUMC, Dept of Medical Microbiology. The plucked eyebrow hair samples were obtained with written permission from renal transplant patients visiting the Dermatology outpatient clinic of the LUMC.
The 50 µl PCR reactions consisted of 1× GeneAmp PCR buffer (15 mM Tris-HCl [pH 8,0], 50 mM KCl, 3,6 mM MgCl, 0,3 mM of each dNTP, 15 pmol of each primer, 7,5 pmol probe and 2 U of AmpliTaq Gold polymerase (Applied Biosystems). Real-time PCR was performed in the iCycler (Biorad) and cycle conditions are 9′ at 95°C, followed by 50 cycles of amplification (94°C for 1 min. and 65°C for 1 min.). TSV copy number was calculated against a plasmid titration series of pUC19-TSV included in each PCR assay that contains the full length TSV genome cloned in XbaI. Cell number was calculated with a PCR specific for the beta-actin gene, which was run in parallel on a dilution series of human genomic DNA (Promega). The sensitivity of each TSV PCR was found to be between 1–10 TSV genome copies.
The cerebrospinal fluids, blood plasmas and respiratory samples with proven JCV-, BKV-, KIV- or WUV-positivity used for validation of the developed TSV-specific PCRs were selected from clinical samples routinely send for viral diagnosis to the LUMC, Dept of Medical Microbiology. The plucked eyebrow hair samples were obtained with written permission from renal transplant patients visiting the Dermatology outpatient clinic of the LUMC.
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