A QUANTITATIVE REAL TIME PCR ASSAY FOR DETECTING BK VIRUS IN SERUM, PLASMA AND URINE

BK is a non-enveloped virus in the Polyomavirus family, closely related to SV40 and JC virus. Primary infection with BK generally occurs during childhood without specific symptoms, and is widespread in the population, with approximately 80% of adults infected globally. The virus remains latent in the urogenital tract, but can become reactivated. Asymptomatic reactivation and sporadic shedding of BK virus in urine can happen spontaneously in immunocompetant patients. � BK virus has been associated with hemorrhagic cystitis in bone marrow transplant patients, as well as with ureteral stenosis and transplant-associated nephropathy in patients receiving kidney transplants. BK virus allograft nephropathy (BKVAN) is distinguished by persistent graft dysfunction, often resulting in graft loss. BKVAN is seen in up to 8% of kidney transplant recipients, frequently targeting renal epithelial tubules. New intensive immunosuppressive regimens are considered a risk factor for BKVAN. Detection of BK has traditionally been done using culture, which is slow, or by electron microscopy, which can be rapid but displays low sensitivity. The use of quantitative real time PCR for BK can provide a valuable tool for the clinician in diagnosing BKVAN as well as monitoring the patient's response to treatment. � We validated a quantitative real time PCR assay on the HT7900 Sequence Detection System (Applied Biosystems) to detect BK virus in serum, plasma and urine. This assay is in an ASR format, and targets the viral VP1 gene using a primer and Eclipse hybridization probe set from Nanogen/Epoch Biosciences. With each new lot of Taq master mix or BK ASR reagent, a new standard curve is generated using a lyophilized plasmid clone, and this curve is stored for quantitation of subsequent assays. Prior to extraction, an exogenous internal control is added to the sample, providing the ability to detect PCR inhibition and extraction failures. To limit the risk of contamination, the assay is run in a 96-well, closed tube format, using UNG and dUTP in the master mix. Accuracy was evaluated by comparing the results of this assay with those obtained using a TaqMan assay performed at another reference lab. The reportable range of the assay is 390 to 390,000,000 copies/mL. Data for precision, linearity and specificity will also be presented.