title: Impact of genomic sequence variability on quantitative PCR assays for diagnosis of polyomavirus BK infection.
abstract: Knowledge of polyomavirus BK (BKV) genomic diversity has greatly expanded. The implications of BKV DNA sequence variation for the performance of molecular diagnostic assays is not well studied. We analyzed 184 publically available VP-1 sequences encompassing the BKV genomic region targeted by an in-house quantitative hydrolysis probe-based PCR assay. A perfect match with the PCR primers and probe was seen in 81 sequences. One Dun and 13 variant prototype oligonucleotides were synthesized as artificial targets to determine how they affected the performance of PCR. The sensitivity of detection of BKV in the PCR assay was a function of the viral genotype. Prototype 1 (BKV Dun) could be reliably detected at concentrations as low as 10 copies/μl. However, consistent detection of all BKV variants was possible only at concentrations of 10,000 copies/μl or higher. For BKV prototypes with 2 or more mismatches (representing genotype IV, genotype II, and genotype 1c strains), the calculated viral loads were 0.57 to 3.26% of the expected values. In conclusion, variant BKV strains lower the sensitivity of detection and may have a substantial effect on quantitation of the viral load. Physicians need to be cognizant of these effects when interpreting the results of quantitative PCR testing in transplant recipients, particularly if there is a discrepancy between the clinical impression and the measured viral load.
authors.affiliation: Division of Transplant Pathology, University of Pittsburgh, Department of Pathology, E737 UPMC-Montefiore Hospital, 3459 Fifth Ave., Pittsburgh, PA 15213, USA. randhawapa@upmc.edu
authors.forename: P
authors.initials: P
authors.lastname: Randhawa
authors.forename: J
authors.initials: J
authors.lastname: Kant
authors.forename: R
authors.initials: R
authors.lastname: Shapiro
authors.forename: H
authors.initials: H
authors.lastname: Tan
authors.forename: A
authors.initials: A
authors.lastname: Basu
authors.forename: C
authors.initials: C
authors.lastname: Luo
mesh_terms: D001739:BK Virus / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D027601:Polyomavirus Infections / Q000175:diagnosis* / Q000821:virology; D012680:Sensitivity and Specificity; D019562:Viral Load; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes