Performance Evaluation of TaqMan Probe Method for BK Virus DNA Quantification by Real-time Polymerase Chain Reaction

Chung, Hee Young; Kim, Yoo Li; Hwang, Kyung Ah; Choi, Byung Hoo; Park, Sook Ja; Sung, Heung Sup; Kim, Mi Na

Abstract

BACKGROUND: We evaluated the performance of a newly developed real-time polymerase chain reaction (PCR) method using TaqMan probe (TP) and internal control (IC) for quantitation of BK virus (BKV) DNA.
METHODS
PCR primers and TP were targeted for the VP1 of BKV and 300 bp-region of VP1 was cloned to prepare a standard DNA. Threshold cycles (Ct) of IC was set at 33+/-3. The recovery rates, precision, linearity, and limit of detection (LOD) were measured using the standard DNA. To correlate TP with previous hybridization probe (HP) method, Ct of those were compared using 35 HP-positive and 15 HP-negative specimens, and the interpretation agreement was analyzed in 63 consecutive clinical specimens including 32 urines and 31 plasmas. Fifty-three53 specimens measured for IC were analyzed for positive rates and levels of BKV according to Ct of IC.
RESULTS
The average recovery rate was 101.1% and intra-assay and inter-assay coefficiency variations were 0.017~0.059 and 0.036, respectively, with the specimens of 3 log/mL, and 0.041~0.063 and 0.045, respectively, with the specimens of 6 log/mL. LOD was 183 copies/mL and linearity range was 2.7 log- 12 log/mL. Ct of TP were correlated with those of HP with the function of y=0.8912x+0.3164 (R2=0.9062). Among 63 clinical specimens, 16 were positive in TP and 12 were positive in HP with an agreement of 90.4%. Ct of IC were over 36 in 31 specimens (22 urines and 9 plasmas), of which BKV DNA was much higher in 7 (22.5%) BKV-positive specimens (5.9+/-1.7 log/mL) than in 4 (18.1%) BKV-positive specimens (3.9+/-1.0 log/mL) of 22 having Ct of IC < or =36.; 5.9+/-1.7 vs. 3.9+/-1.0 log/mL.
CONCLUSION
TP warrants to be a reliable method for quantification of BKV. IC seemed to be essential to differentiate false-negative results or underestimation of BKV in clinical specimens, especially in urine.