BKV-DNA and JCV-DNA co-quantification assay to evaluate viral load in urine and serum

• Published in: Molecular biotechnology 2005-05, Vol.30 (1), p.1-8
• Main Authors: Merlino, Chiara, Bergallo, Massimiliano, Daniele, Roberta, Ponzi, Alessandro Negro, Cavallo, Rossana
• Format: Article
• Language: English
• Subjects: Antigen T (large); BK Virus - genetics; Deoxyribonucleic acid; DNA; DNA, Viral - blood; DNA, Viral - urine; Female; Humans; Immunosuppression; Infection; JC Virus - genetics; Kidney transplantation; Kidney Transplantation - adverse effects; Kidney Transplantation - methods; Male; Nephritis; Nephritis - complications; Nephritis - therapy; Nephritis - virology; Nephropathy; Polymerase chain reaction; Polymerase Chain Reaction - methods; Polyomavirus; Polyomavirus Infections - blood; Polyomavirus Infections - etiology; Polyomavirus Infections - urine; Primers; Reproducibility of Results; Running; Sensitivity and Specificity; Studies; Transplants & implants; Tumor Virus Infections - blood; Tumor Virus Infections - etiology; Tumor Virus Infections - urine; Urine; Viral Load - methods; Viruses
• ISSN: 1073-6085; 1073-6085; 1559-0305
• DOI: 10.1385/mb:30:1:001

Infections from human polyomaviruses BK and JC (BKV and JCV) occur independently, but concomitant infections and the simultaneous persistence of both viruses have been observed in renal transplant recipients. Several studies have disclosed a correlation between BKV and interstitial nephritis in renal transplant recipients, and an association between JCV and some cases of nephropathy has recently been hypothesized. This article describes the development of a semiquantitative-nested polymerase chain reaction (PCR) assay to simultaneously detect BKV and JCV viral load in urine and serum. The first-round amplification step uses primers that amplify a 385-bp DNA fragment from the "large T antigen" region of both viruses. Samples testing positive in the first step are then run in the second step. In the second-round amplification, different inner primers are used to separately quantify BKV-DNA and/or JCV-DNA. The assay offers several advantages including: (1) rapid submission of clinical samples to screening; (2) verification of the absence of Taq polymerase inhibitors with the use of an internal control; (3) a sensitivity threshold of 10 copies/reaction; and (4) assay running is less labor intensive, cheap, and easy to perform. The assay may be easily used to monitor viral loads versus baseline levels in urine and serum samples from renal transplant recipients to detect those at risk of BKV- or JCV-related nephropathy, and to monitor their response to immunosuppression reduction therapy if it occurs.