Development of a Bak gene based standard curve for absolute quantification of BK virus in real time quantitative PCR assay and noninvasive diagnosis of BK virus nephropathy in kidney allograft recipients

Abstract

BackgroundBK virus nephropathy (BKVN) is a frequent and serious post-transplant complication and undermines realization of the full benefits of kidney transplantation. We developed a Bak amplicon-based standard curve for absolute quantification of BKV VP1 mRNA copy number in the real time quantitative PCR (RT-qPCR) assay and investigated the performance characteristics of this novel assay. MethodsWe determined analytical specificity, sensitivity, and precision of our 73 bp mouse Bak amplicon based standard curve for absolute quantification of BKV VP1 mRNA in RT-qPCR assays. The diagnostic accuracy of the Bak standard curve in the RT-qPCR assay for the noninvasive diagnosis of BKVN in human kidney allograft recipients was investigated by quantification of BKV VP1 mRNA copy number in 192 urine samples matched to 192 kidney allograft biopsies from 155 unique kidney allograft recipients. Intraclass correlation coefficients (ICC) were calculated for the threshold cycles (Ct) and BKV VP1 mRNA copy number observed in the RT-qPCR assay with the Bak standard curve or the BKV standard curve. ResultsPerformance characteristics of the Bak amplicon-based RT-qPCR assay were exceptional with a slope of −3.291, Y-intercept of 38.60, R2 value of 1.00, efficiency of 101% and error of 0.014. Amplification was specific for the Bak amplicon. Intra assay standard deviation (SD) was 0.08 or less and inter assay SD was 0.11 or less for 31 cycles or less of amplification of the Bak amplicon. Receiver operating characteristic (ROC) curve analysis of BKV VP1 mRNA copy number in 192 biopsy matched urines yielded an area under the ROC of 0.982 (95% CI, 0.964 to 0.999, P < 0.0001) for discriminating patients with BKVN biopsies from patients without BKVN biopsies. The striking identity in the measurement of BKV VP1 mRNA copy numbers in the Bak amplicon-based RT-qPCR assay and in the BKV amplicon-based RT-qPCR assay was shown by an ICC of 1.00 when the Cts were compared, and an ICC of 0.99 when the log10 BKV VP1 mRNA copy numbers were compared. ConclusionsBak standard curve for absolute quantification of BKV VP1 mRNA copy number in the RT-qPCR assay demonstrated high efficiency, short and long-term precision and analytical specificity. BKVN was diagnosed with high accuracy. Our new findings, viewed in the light of our earlier demonstration that absolute quantification of a panel of mRNAs encoding immunoregulatory proteins is feasible with the Bak amplicon-based RT-qPCR assays, suggest that the Bak standard curve could serve as a universal calibrator for absolute quantification of transcripts in RT-qPCR assays and help reduce the workload, costs and eliminate contamination of genes of interest by repeated amplification of gene specific standard curves.