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J Virol Methods. 2006 Jul;135(1):32-42. Epub 2006 Mar 9.

Real-time, quantitative PCR assays for the detection of virus-specific DNA in samples with mixed populations of polyomaviruses.

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1
Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.

Abstract

Mixtures of polyomaviruses can be present in the central nervous system, the gastrointestinal tract, the genitourinary tract, blood, and urban sewage. We have developed 12 primer/probe sets (four per virus) for real-time, quantitative PCR assays (TaqMan) that can specifically detect BKV, JCV, and SV40 genomes present in mixtures of these viruses. The specificities of these primer/probe sets were determined by evaluating their level of interaction with the DNA from other polyomaviruses and their ability to estimate the number of copies of homologous viral DNA in blinded samples of defined mixtures of three polyomaviral DNAs. Three early region and three late region primer/probe sets determined, within a two-fold range, the number of copies of their respective DNAs. Four sets of SV40 primer/probes also detected 1.1-2.4 copies of SV40 DNA per COS-1 cell, cells estimated to contain a single copy of SV40 DNA. Three JCV primer/probe sets detected 3.7-4.2 copies per cell of JCV DNA in the JCV-transformed cell line M1-HR, cells estimated to contain between 0.5 and 1 copy of the JCV genome. We suggest that the virus-specific primer/probe sets in this study be considered sufficiently characterized to initiate the quantification of polyomavirus DNA in biological samples.

PMID:
16527364
DOI:
10.1016/j.jviromet.2006.01.018
[Indexed for MEDLINE]
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