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Journal of Clinical Microbiology, October 2000, p. 3689-3695, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Rapid Quantification and Differentiation of Human Polyomavirus DNA in Undiluted Urine from Patients after Bone Marrow Transplantation

Stefan S. Biel,1 Thomas K. Held,2 Olfert Landt,3 Matthias Niedrig,1 Hans R. Gelderblom,1 Wolfgang Siegert,2 and Andreas Nitsche2,*

Robert Koch-Institut,1 Klinik für Innere Medizin m.S. Hämatologie und Onkologie, Charité Campus Virchow-Klinikum, Humboldt Universität zu Berlin,2 and TIB Molbiol,3 Berlin, Germany

Received 3 February 2000/Accepted 24 March 2000

A combined PCR assay was developed for the detection and typing of human polyomavirus (huPoV) in clinical samples, consisting of (i) a qualitative seminested PCR assay (snPCR) to discriminate between huPoV BK and JC and (ii) a high-throughput, quantitative TaqMan PCR assay (TM-PCR) for the general detection of huPoV. The TM-PCR detects huPoV DNA in a linear range from 107 to 101 copies per assay. In reproducibility runs, the inter- and intra-assay variabilities were <= 60 and <= 50%, respectively. The snPCR assay uses a set of four primers for the same region of the BK and JC viral genomes. In the first round of amplification, two general primers were used; in the second round, one of these general primers and two additional, BK- or JC-specific primers were used simultaneously to produce amplicons of different sizes specific for BK virus (246 bp) and JC virus (199 bp), respectively. We tested different urine dilutions in order to determine the inhibitory effects of urine on PCR amplification. Furthermore, we compared the use of native urine with DNA purified by different preparation procedures. Our results show, that a 1:10 dilution of the urine led to complete reduction of the amplification inhibition found with 6% of undiluted urine samples. In a clinical study including 600 urine specimens, our assay turned out to be fast, cheap, and reliable in both qualitative and quantitative aspects.

* Corresponding author. Mailing address: Charité Campus Virchow-Klinikum, Klinik für Innere Medizin m.S. Hämatologie und Onkologie, Forschungshaus 37, R. 2.0303, Augustenburger Platz 1, D-13353 Berlin, Germany. Phone: 49-30-45059-398. Fax: 49-30-45043-929. E-mail: andreas.nitsche{at}charite.de.

Journal of Clinical Microbiology, October 2000, p. 3689-3695, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Dumonceaux, T. J., Mesa, C., Severini, A. (2008). Internally Controlled Triplex Quantitative PCR Assay for Human Polyomaviruses JC and BK. J. Clin. Microbiol. 46: 2829-2836 [Abstract] [Full Text]  
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