Development and validation of a quantitative real time PCR assay for BK virus

Midori Mitui, N. Kristine Leos, Damon Lacey, Christopher Doern, Beverly B. Rogers, Jason Y. Park

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Several studies have shown that BK viral load in plasma and urine are reliable markers for the detection of BK virus associated nephropathy (BKVAN) in renal transplant patients. We developed a quantitative real time PCR assay based on TaqMan technology for the measurement of BK viral load in plasma and urine. Considering the high similarity of the nucleotide sequence of the BK virus (BKV) with the JC virus (JCV), we designed this assay to specifically amplify BKV. We determined the viral DNA recovery rate on manual (QIAGEN's QIAamp DNA Blood Mini Kit) and automated (BioMerieux's NucliSENS EasyMAG) extraction methods. The comparison showed a higher viral DNA recovery rate on the automated extraction (61-76% in plasma and 52-65% in urine) as compared to the manual method (49-52% in plasma and 33-56% in urine). Quantitation of the viral load was performed using an external standard curve that was constructed with serial dilution of a plasmid containing the full length of the BKV genome. Commercially available quantitative BKV standards showed good correlation with the plasmid standard. The reproducibility of the assay was determined based on the Ct values of the amplified products as well as in BK copies per milliliter of sample. This assay is linear over a 7 log range (10 to 1×107 copies per reaction), no cross-reactivity was detected with the closest-related polyomavirus JCV, as well as other viruses that may be found in immunocompromised patients, and human genomic DNA. The limit of detection of the assay is 300 copies per milliliter in both plasma and urine and the limit of quantitation is 1000 copies per milliliter using the NATtrol BK Virus Linearity Panel (ZeptoMetrix). This real time PCR assay provides a reliable and sensitive method for the quantitation of BKV in plasma and urine samples.

Original languageEnglish (US)
Pages (from-to)230-236
Number of pages7
JournalMolecular and Cellular Probes
Volume27
Issue number5-6
DOIs
StatePublished - Oct 1 2013

Keywords

  • BK nephropathy
  • BK virus
  • Infectious disease
  • Nucleic acid extraction
  • Real time PCR

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Development and validation of a quantitative real time PCR assay for BK virus'. Together they form a unique fingerprint.

  • Cite this