Quantitative real-time polymerase chain reaction detection of BK virus using labeled primers.
BACKGROUND
BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease.


OBJECTIVE
To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers.


METHODS
Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization).


RESULTS
Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 10(1) to 10(7) copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R(2) value of 0.96 between the 2 assays.


CONCLUSIONS
Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.
Gu
Zhengming
Z
Departments of Pathology, St Jude Children's Research Hospital, Memphis, Tennessee 38103, USA.
Pan
Jianmin
J
Bankowski
Matthew J
MJ
Hayden
Randall T
RT
D001739:BK Virus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D006801:Humans; D057089:Pathology, Molecular / Q000379:methods; D027601:Polyomavirus Infections / Q000175:diagnosis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000175:diagnosis*; D019562:Viral Load
D017931:DNA Primers; D004279:DNA, Viral