title: A real time genotyping PCR assay for polyomavirus BK.
abstract: BACKGROUND
  Polyomavirus BK (BKV) may cause nephropathy in renal transplant recipients and hemorrhagic cystitis in bone marrow recipients. We developed real-time PCRs (RT-PCR) to determine easily and rapidly the different BKV genotypes (BKGT) (I-IV).
  
  
  METHODS
  On the VP1 gene a duplex of RT-PCRs was developed and validated to differentiate the four main BKGT. 212 BKV positive samples (21 plasma, 191 urine) were tested with these specific PCRs. Of these 212 samples, 55 PCR results were additionally confirmed by sequencing a VP1 gene fragment (nucleotide 1630-1956).
  
  
  RESULTS
  For every genotype, a highly specific, precise and internally controlled assay was developed with a limit of detection of log 3 copies per ml. In 18 (8.5%) of these samples genotyping was not successful due to a low viral load. By sequence analysis, the genotype of 46 out of 55 and 2 out of 4 samples with double infection could be confirmed.
  
  
  CONCLUSIONS
  This study describes RT-PCRs for detection of the main BKGT. It proved to be rapid, cheap and sensitive compared to sequencing. Double infections can also be detected. This method will be of value to investigate the role of BKV infection in relation to the genotype.
authors.affiliation: Department of Medical Microbiology, Division of Clinical Virology, The University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. Electronic address: l.gard@umcg.nl.
authors.forename: Lilli
authors.initials: L
authors.lastname: Gard
authors.affiliation: Department of Medical Microbiology, Division of Clinical Virology, The University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
authors.forename: Hubert G M
authors.initials: HG
authors.lastname: Niesters
authors.affiliation: Department of Medical Microbiology, Division of Clinical Virology, The University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
authors.forename: Annelies
authors.initials: A
authors.lastname: Riezebos-Brilman
mesh_terms: D001739:BK Virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D036022:Capsid Proteins / Q000235:genetics*; D003365:Costs and Cost Analysis; D060005:Genotyping Techniques / Q000191:economics / Q000379:methods*; D006801:Humans; D027601:Polyomavirus Infections / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014412:Tumor Virus Infections / Q000821:virology; D014773:Virology / Q000191:economics / Q000379:methods*
chemical_list: D036022:Capsid Proteins; C062481:VP1 protein, polyomavirus
keywords: Genotyping; Polyomavirus BK; Real time PCR