Comparison of PCR-southern hybridization and quantitative real-time PCR for the detection of JC and BK viral nucleotide sequences in urine and cerebrospinal fluid.
The presence of the human polyomaviruses JCV and BKV in immunocompromised patients can lead to lethal diseases and conditions including progressive multifocal leukoencephalopathy (PML), interstitial nephritis, hemorrhagic cystitis, and kidney allograft rejection. Typically, detection of JCV and BKV in clinical samples has employed standard PCR amplification for viral nucleotide sequences, with subsequent confirmation for viral genome specificity of PCR products by southern blot hybridization. Here, we directly tested a validated PCR-southern protocol with a TaqMan real-time PCR protocol (Applied Biosystems) to assay clinical samples of urine and cerebrospinal fluid. We found equal specificity and sensitivity with both methods. However, real-time allowed for absolute viral-genome quantitation without the use of radionucleotides and was performed more rapidly, in as little as 24 h. Such advantages are important to consider in the effort to establish international standardization of controls for the detection of JCV and BKV, which would aid in screening confidence and the reliable assessment of anti-viral therapies.
Ryschkewitsch
Caroline
C
Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 36 Convent Drive, Bethesda, MD 20892-4164, USA.
Jensen
Peter
P
Hou
Jean
J
Fahle
Gary
G
Fischer
Steven
S
Major
Eugene O
EO
D001739:BK Virus / Q000235:genetics / Q000302:isolation & purification*; D026501:Blotting, Southwestern; D004279:DNA, Viral / Q000032:analysis; D006801:Humans; D007577:JC Virus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D027601:Polyomavirus Infections / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000652:urine; D014412:Tumor Virus Infections / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000652:urine
D004279:DNA, Viral