Real Time PCR Assays Compared for Viral Quantification

The performance of three real time polymerase chain reaction assays has been compared for the quantification of polyomavirus BK.

Reactivation of latent polyomavirus BK is associated with nephropathy (PVAN) after renal transplantation and the BK viral load determinations are a highly sensitive and specific method for predicting risk for PVAN.

At the Albany Medical Center Hospital (NY, USA) a team of investigators collected 100 anticoagulated plasma specimens from 69 renal transplant patients which were analyzed with three real-time PCR (rt-PCR) assays for BK viral load determination. The rt-PCR assays were evaluated in terms of linearity, dynamic range, and accuracy, against a conventional PCR with primers and probe targeting the large T antigen (TOR) (Integrated DNA Technologies, Coralville, IA, USA).

The two commercial rt-PCR assays used were the MultiCode-RTx BK (MC-RTx) virus Analyte Specific Reagents (ASR) from Luminex (Austin, TX, USA), and the MGB-Alert BKV ASR (MGB) from Epoch Biosciences (Princeton, NJ, USA). A laboratory developed assay (LDA) was also evaluated. Of the 100 specimens from 69 patients sent to the laboratory for routine polyomavirus measurements by the TOR and analyzed with all three rt-PCR assays, 76 were defined as true positive for BKV DNA and no samples were inhibitory for PCR.

The MC-RTx assay was positive for all 76 specimens whereas MGB was positive for 64 and LDA was positive for 55. The viral load range for these samples was from1.71 to 7.55 log10 copies/mL, while the median viral load was 3.01 log10 copies/mL. BKV DNA was not detected by any method in the true negative specimens. The sensitivities of the assays was 100% for MC-RTx, 84% for MGB and 72% for LDA and the specificities were 100% for all three assays.

The authors concluded that all three real-time PCR assays are reliable for measuring BKV loads for the identification of patients at risk for PVAN. These assays demonstrated superiority over the TOR with regards to precision. MC-RTx appears to have enhanced sensitivity and better performance among divergent strains of BK. The study was published on December 27, 2012, in the Journal of Clinical Virology.

Related Links:
Albany Medical Center Hospital
Integrated DNA Technologies
Luminex