Quantitative real-time PCR assay for detection of human polyomavirus infection

Journal of Virological Methods 135 (2006) 207–213

Quantitative real-time PCR assay for detection of human polyomavirus infection Amal Elfaitouri a , Anna-Lena Hammarin b , Jonas Blomberg a,∗ a

Section of Virology, Department of Medical Sciences, Uppsala University Hospital, S-571-85 Uppsala, Sweden b Swedish Institute for Infectious Disease Control (SIIDC), Solna, Sweden Received 24 October 2005; received in revised form 9 March 2006; accepted 16 March 2006 Available online 4 May 2006

Abstract The human polyomaviruses BK (BKV) and JC (JCV) affect immunosuppressed patients and are associated with urogenital tract (BKV) and CNS disorders (JCV) and in humans, the pathogenic role of the rhesus monkey virus, Simian virus 40 (SV40), is uncertain. These three viruses have somewhat overlapping tissue pathogenicity and detection of all three polyomaviruses is desirable. A broadly targeted, simple, single tube real-time degenerated quantitative PCR (QPCR) technique for detection of JCV, BKV and SV40 DNA was developed. To avoid false positive results, due to contamination with commonly used SV40 T-antigen plasmids, a conserved region of the VP2 gene was targeted. Down to 1–10 copies of target DNA per PCR reaction were detected. The QPCR was compared with a nested PCR on 41 clinical samples (urine, serum and plasma): 24 (58.5%) tested positive by nested PCR, whereas 31 (75.6%) were positive with QPCR. One CSF sample, from a patient with progressive multifocal leukoencephalopathy, was negative with the nested PCR but determined as positive by QPCR. Sera from 24 blood donors were negative with QPCR. The QPCR described had a high sensitivity. Its specificity was confirmed sequencing. The QPCR is simple to perform and is valuable for diagnosis of polyomavirus infection. © 2006 Elsevier B.V. All rights reserved. Keywords: Broadly targeted PCR; Real-time PCR; QPCR; Polyomavirus; JC virus; BK virus (BKV); Simian virus 40 (SV40)

1. Introduction The human polyomaviruses BK (BKV) and JC (JCV) and Simian virus 40 (SV40) are species of the genus polyomavirus within the Polyomaviridae, a family of non-enveloped DNA viruses with an icosahedral capsid of 45 nm in diameter. The capsid contains a closed, circular, double-stranded DNA genome with an average length of 5 kb. The primate polyomavirus genome is divided into an early region, a late region and a noncoding control region (NCCR). The early region encodes the small and large T-antigens (tAg and TAg), and the late region encodes the viral capsid proteins VP1, VP2 and VP3, as well as the agnoprotein. The NCCR contains efficient promotor and enhancer sequences essential for replication of viral DNA. Plasmids carrying the early region of SV40 are frequently used in virological laboratories. Moreover, the TAg is responsi-

∗

Corresponding author. Tel.: +46 18 611 55 93; fax: +46 18 55 10 12. E-mail address: [email protected] (J. Blomberg).

0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.03.006

ble for the ability of polyomaviruses to transform cells both in vivo and in vitro (Gottlieb and Villarreal, 2001). The genomes of the three polyomavirus JCV, BKV, and SV40 share an overall identity of 70–72%. Humans are natural hosts for BKV and JCV and both viruses are widely distributed (70–100%) in the human population (de Bruyn and Limaye, 2004; Major et al., 1992). The primary infection and persistence of BKV and JCV are generally asymptomatic in healthy individuals; however, reactivation of these viruses may occur in immunosuppressed individuals (Bogdanovic et al., 1992; Norkin, 1982). Reactivation of BKV is primarily associated with urinary tract disorders, mainly in bone marrow (Arthur et al., 1986) and renal transplant patients (de Bruyn and Limaye, 2004; Limaye et al., 2001). Reactivation of JCV may cause progressive multifocal leukoencephalopath (Major et al., 1992). SV40 is a macaque polyomavirus suggested to have been transferred to humans via contaminated polio vaccines during the 1950s (Carbone et al., 1997). SV40, BKV and JCV are potent oncogenic viruses and all three can induce, or are associated with, tumour development

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in laboratory animals (Jha et al., 1998; Reiss and Khalili, 2003; Tognon et al., 2003). However, the association of polyomavirus with human malignancy is contradictory. Although the involvement of SV40 in human mesotheliomas is reported; reviewed in (Carbone et al., 2003; Garcea and Imperiale, 2003; Klein, 2000); the specificity of this finding is debated (Lopez-Rios et al., 2004). One problem is the risk of false positive PCR results due to contamination with common laboratory plasmids containing fragments of the SV40 genome, usually the TAg gene. Epidemiological studies of humans exposed to SV40 contaminated poliovirus vaccines fail to demonstrate an association between SV40 and cancer (Tognon et al., 2004). Genomes of BKV and JCV have been detected in human tumours. Besides mesotheliomas, primarily in brain tumours (Croul et al., 2003; Rollison et al., 2005), JCV DNA has been found in upper digestive tract carcinomas (Del Valle et al., 2005). According to serological data, BKV generally infects at age 3–4 years, whereas JCV generally infects at an age of 10–14 years (Arthur and Shah, 1999). SV40 infection seems less common (Shah, 2004), although limited data indicate that the age at SV40 infection in humans may be closer to that of BKV than for that of JCV (Lundstig et al., 2005; Vanchiere et al., 2005). The predilection of JCV for brain and BKV for the urinary tract is not absolute. In a few cases, BKV has been detected in patients with CNS disorders (Bratt et al., 1999; Jorgensen et al., 2003) and JCV has been reported in allograft nephropathy in renal transplant recipients in the absence of BKV (Kazory et al., 2003). However, SV40 can also cause polyomavirus nephropathy (PVN) (Milstone et al., 2004) and hemorrhagic cystitis (HC) in children after bone marrow transplantation (BMT) (Comar et al., 2004). Therefore, BKV, JCV, and to some extent SV40 may have overlapping tissue pathogenicity profiles and a PCR which detects DNA of all three polyomaviruses in clinical samples is therefore desirable. A broadly targeted quantitative real-time PCR (QPCR) technique, based on the TaqMan® technology for amplification of BKV, JCV, and SV40 DNA, was developed. Degenerate primers that detect a conserved region of the VP2 gene of BKV, JCV, and SV40 were analyzed with QPCR: this region was detected with a bioinformatic analysis of all primate polyomaviral sequences available in GenBank. DNA sequencing ascertained the type of polyomavirus detected in the sample. 2. Materials and methods 2.1. Clinical samples and control material Forty-one anonymous samples from bone marrow and kidney transplant patients (32 urine, 7 sera and 2 plasma) and one CSF sample from a patient with progressive multifocal leukoencephalopathy were obtained from the Swedish Institute for Infectious Disease Control (SIIDC). A further 24 sera from anonymous blood donors from the Blood Bank of Uppsala Academic Hospital served as negative controls, and two urine

samples with a previously determined content of BKV and JCV served as positive controls. 2.2. Nested PCR for detection of BKV and JCV DNA The 41 samples from transplant patients were examined with a previously described nested PCR for detection of BKV and JCV DNA (Bogdanovic et al., 1994). It also detects SV40 (Hammarin, unpublished observation). This technique uses boiled samples instead of DNA extraction. Thus, the comparison with this PCR technique involved also the mode of DNA preparation. The type of virus was established in the positive samples by restriction enzyme cleavage of amplimers and gel electrophoresis. 2.3. DNA extraction DNA was prepared from 200 ␮L serum, plasma and CSF samples with the QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany), according to the instructions of the manufacturer. The DNA extract is normally obtained in 200 ␮L of buffer. For the CSF sample, the elution step was with 50 ␮L buffer AE, resulting in a higher DNA concentration according to the instructions of the manufacturer. Theoretically, this concentration also produces a higher concentration of PCR inhibitors (see Section 4). DNA was prepared from urine samples with the QIAamp® viral RNA Mini Kit (also optimised for DNA preparation from urine; Qiagen, Hilden, Germany), according to the manufacturer’s instructions. 2.4. Design of primers and probe for JCV, BKV and SV40 Complete or partial genomes from 121 BKV, 277 JCV, 19 SV40, 1 Rhesus macaque polyoma, available in GenBank in August 2003, were aligned with BLAST (http://www. ncbi.nlm.nih.gov/blast/), with the reference strain of human BK virus, strain Dunlop (V01108.1), as query.A baboon polyoma sequence was later included in the analysis (Cantalupo et al., 2005). The BLAST alignments were examined by the ConSort© program for conserved stretches (Blomberg J., unpublished). A suitably conserved 113 base pair stretch was detected in the VP2 gene (Fig. 1). Degenerate primers and probe, forward primer BJS-FP, reverse primer BJS-RP, and probe BJS-P were designed. Their sequences and Tm -values are presented in (Table 1). After evaluation, primers and probe were designed with the on-line software OligoAnalyzer 3.0 (http://207.32.43.70/ biotools/oligocalc/oligocalc.asp). Primers were ordered from Thermo Hybaid, Interactiva Division (Ulm, Germany). The probe was ordered via MedProbe (Lund, Sweden) from Eurogentec (Seraing, Belgium). TagMan® probe was labelled with the fluorescent reporter dye 6-FAM (6-carboxyfluorescein) at the 5 -end and quenching dye TAMRA at the 3 -end. During the optimisation, various concentrations of primers and probe were tested. PCR products were initially subjected to 2% agarose gel

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Fig. 1. The results of the conservation analysis, from the ConSort© program, of the BLAST results from alignments of complete or partial genomes from 121 BKV, 277 JCV, 19 SV40, 1 Rhesus macaque polyoma available in GenBank in August 2003 with the reference strain of human BK virus, strain Dunlop (V01108.1), as query. The output from the ConSort© program shows, first, the most common variant for each position, second, the less common variants in decreasing order. Insertions and deletions are indicated. Capital letters in the alternative nucleotides signify variants present in more than 2% of all aligned sequences. Capital letters in the consensus sequence row indicate presence of a conserved stretch, which may or may not be suitable for primer and probe construction. The positions of highly conserved stretches were selected for primers and probe such as: BJS-FP (p648–667), BJS-P (p687–718) and BJS-RP (p741–760).

electrophoresis to verify that fragments of correct length were amplified. 2.5. Real-time QPCR system Reactions were amplified in a Corbett Research RotorGeneTM 3000 Real-Time Thermal cycler (Corbett Research, Mortlake, Australia); real-time PCR data was analysed with the accompanying Rotorgene Real-time Analysis Software. In most cases, amplifications were in a final volume of 25 ␮L containing 1 ␮L of template: this volume was chosen to minimize possible inhibition, although in a few instances 5 ␮L of DNA extract were also used with no obvious negative effects. Further, 2.5 ␮L of 10× Gold PCR buffer® without Mg2+ and final concentrations of 200 ␮M dNTPs (equal mix) (Applied Biosystems, Stockholm, Sweden) and 200 nM primers BJS-FP and BJS-RP, and 150 nM BJS-P probe; 2 mM MgCl2 ; 1.25 units of AmpliTaq Gold DNA polymerase© (Applied Biosystems) were added.

Finally, the volume was adjusted to 25 ␮L with nuclease-free water. Thermal conditions were 95 ◦ C for 10 min, followed by 45 cycles of denaturation at 95 ◦ C for 45 s, annealing at 49 ◦ C for 30 s and extension at 72 ◦ C for 30 s. In each QPCR run, a non-template control was included to monitor possible contamination. The DNA from clinical samples was extracted as described above (Section 2.3) and tested in duplicate with the QPCR assay. 2.6. Specificity and quantification of QPCR Twenty-four serum samples from anonymous blood donors from Uppsala, Sweden, were used to test the specificity of the QPCR. Ten-fold dilutions of SV40 plasmid DNA, range, 105 to 101 copies/␮L, were used for calculation of approximate copy numbers (gene equivalents/mL) and for standardization of QPCR. The SV40 DNA plasmid, originally from the American Type Culture Collection (ATCC® Cat. No. 45019), was kindly

Table 1 Oligonucleotide primers and probe used for VP2 QPCR Primers and probes

Sequence 5 –3a

Degeneracy

Estimated Tm (◦ C)b

BJS-FPc BJS-RPd BJS-Probee

GGG GAC CTA RTT GCY AST GT GCA ASR GAT GCA AKT TSMAC ACW GGA TTT TCA GTR GCT GAA ATT GCT GCT GG

8 32 4

56.7 (54.1–59.3) 54.0 (50.3–58.0) 63 (62.1–64.0)

a b c d e

IUPAC ambiguity codes. R = AG; Y = CT; K = GT; S = CG; W = AT; M = AC. The melting temperatures were estimated with Oligo Analyzer 3.0 software. BJS-FP (forward primer). BJS-RP (reverse primer). Reporter dye (6-FAM) at 5 . Quencher dye (TAMRA) at 3 .

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provided by Professor G¨oran Magnusson from the Department of Medical Biochemistry and Microbiology, BMC, Uppsala University, Uppsala, Sweden. The sensitivity was first estimated by end-point titrations, with four observations per dilution step, into the stochastic zone of 1–10 DNA copies per QPCR reaction. Four clinical samples, two positive for JCV and two for BKV, were titrated in 10-fold dilution steps down to complete negativity. The sensitivity also assessed with synthetic JCV (sequence AF396435.1, positions 274–386), BKV (sequence V01108.1, positions 648–760) and synthetic SV40 (sequence J02400.1, positions 585–698) oligonucleotides. (Thermo Hybaid, Interactiva Division, Ulm, Germany).

1 ␮L of PCR reaction (1–3 ng, PCR product 100–200 bp), 1 ␮L of 3.2 pmol BJS-FP or BJS-RP and nuclease-free water. The samples were subjected to 25 cycles of 10 s at 96◦ C, 5 s at 50 ◦ C, 4 min at 60 ◦ C. To purify extension products, ethanol was precipitated in microcentrifuge tubes, according to the instructions of the manufacturer. The samples were then analysed in an ABI PRISMTM 310 Genetic Analyzer® (Applied Biosystems). From the data obtained on the sequences, a BLAST search identified the virus type compared with the GenBank databases. 3. Results 3.1. Identification of conserved portions in VP2 of JCV, BKV and SV40 for design primers and probes

2.7. Sequences of PCR products The 113 nucleotides of the VP2 region were sequenced in amplimers of the seven QPCR positive urine samples, which were tested negative by nested PCR. The BKV and JCV positive urine samples used as positive controls were also sequenced (see Section 2.1). PCR samples were in a total volume of 25 ␮L containing 2.5 ␮L of template, 2.5 ␮L of 10× Gold PCR buffer® without Mg2+ , 200 ␮M dNTPs (equal mix) (Applied Biosystems, Stockholm, Sweden); 200 nM of BJS-FP, BJS-RP primers and 150 nM of BJS-P probe; 2 mM MgCl2 ; 1.25 units of AmpliTaq Gold DNA polymerase© (Applied Biosystems); and nucleasefree water. Amplification reactions were performed by a Corbett Research Rotor-GeneTM 3000 Real-Time Thermal cycler (Corbett Research, Mortlake, Australia). PCR product DNA concentrations were measured by the DyNaQuant 200 fluorometer (Amersham Pharmacia Biotech, Sweden) with calf thymus DNA as a calibrator. The PCR products were subsequently used as templates in 20 ␮L sequencing reactions with the ABI PRISM® BigDyeTM Terminator v2.0 Cycle Sequencing Ready Reaction Kit. For each sequencing reaction, 2 ␮L of Terminator Ready Reaction mix,

The ConSort© analyses of all available primate polyomaviruses in GenBank determined a number of stretches conserved between BK, JC and SV40 viruses. Primers and probe were selected from a conserved 113 bp region based on the VP2 gene of 277 JCV, 121 BKV and 19 SV40 complete or partial genome sequences found by the ConSort© program: their positions are indicated by numbers in Fig. 1. The sequences of the primers and probe were available at GenBank, AF396435.1|JCV (strain #249E; L2001): 274-386, V01108.1|BKV (strain Dunlop): 684–760 and J02400.1|SV40 (strain 776): 585–698 (Fig. 2). The BK-like baboon polyoma virus sequence (Cantalupo et al., 2005) had only one mismatch, in the middle of the reverse primer, in the described primers and probe, rendering uncertainty as to whether this virus would be detected. When designing the oligonucleotide, care was taken to minimize the degree of primer and probe self- and intercomplementarity. For gel electrophoresis, clear bands of the expected size (113 bp) were obtained with 200 nM BJS-FP, BJS-RP and no primer–dimer band. TaqMan probe BJS-Probe was then designed to fit into the system and primers and probe concentration optimisation assays were performed (data not shown).

Fig. 2. Alignment of a selected conserved part of the VP2 gene (113 bp) for the BKV, JCV and SV40 after analysis in the ConSort© program (Fig. 1). Positions of aligned sequences were: AF396435.1|JCV: 274–386, V01108.1|BKV: 648–760 and J02400.1|SV40: 586–698. Primer and probe positions are shown in bold, BJS-FP1-20, BJS-P 40–71 and BJS-RP 94–113. The distinction of BKV from JCV depends on only four nucleotides in the space between primers BJS-P and BJS-RP. Seven nucleotides distinguish SV40 from BKV and JCV (underlined).

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A 200 nM concentration of BJS-FP and BJS-RP primers and 150 nM BJS-Probe were optimal for the QPCR assay. 3.2. Comparison between QPCR and nested PCR for detection of BKV and JCV DNA in clinical samples The presence of BKV and JCV DNA in 41 clinical samples from kidney and bone marrow transplanted patients were analysed with nested PCR, as described earlier (Bogdanovic et al., 1994). Twenty-four of the 41 (58.5%) samples were positive with the nested PCR. These 41 samples were analysed by QPCR without information on the nested PCR results: 31 (75.6%) were positive. Ten were negative in both PCRs and seven were positive with QPCR only. The sequences of the PCR products from the seven positive QPCR samples confirmed the specificity of the QPCR. However, it should be stressed that the distinction of BKV from JCV depended on only four nucleotides in the space between primers BJS-FP and BJS-RP. Seven nucleotides distinguished SV40 from BKV and JCV (underlined in Fig. 2), with this limitation in mind, six out of seven were from BKV and one from JCV. 3.3. JCV DNA detected by QPCR in CSF sample from a progressive multifocal leukoencephalopathy patient Nested PCR (Bogdanovic et al., 1994) is used for the detection of JCV DNA in CSF in several studies (Bogdanovic et al., 1998; Hammarin et al., 1996; Weber et al., 1997). In this study, one CSF sample from a patient with progressive multifocal leukoencephalopathy was tested. It was positive (200 equivalents ␮L of DNA extract) in QPCR. The sample was negative by nested PCR, but confirmed to be positive by another real-time PCR for JCV DNA, as described by (Bossolasco et al., 2005). In this case, the sensitivity was also enhanced by elution of DNA from the QIAprep column with volumes of less than 200 ␮L (50 ␮L) in order to increase the final DNA concentration in the eluate, according to the instructions of the manufacturer. 3.4. Calculation of specificity and sensitivity of QPCR The molecular limit of detection of QPCR was 10 DNA copies of the SV40 plasmid. Serial 10-fold dilutions of synthetic DNA oligonucleotides from JCV, BKV and SV40 ranging from 1 × 100 to 1 × 107 copies were run in QPCR. Standard curves displayed a linear relationship for QPCR assay, with efficiencies of 89% for JCV, 88% for BKV and 71% for SV40 target oligonucleotides. Under these conditions, the limits of detection were 1–10 copies per PCR reaction for each of JCV, BKV and SV40. Results of the amplification curves and standard curve of QPCR for synthetic BKV are shown in Fig. 3A and B. Sensitivity was also studied by end-point titrations, with four observations per dilution step, into the stochastic zone of 1–10 DNA copies per QPCR reaction. Four clinical samples, two positive for JCV and two for BKV, were titrated in tenfold dilution steps down to complete negativity. The result indicated a sensitivity of 1–10 copies per

Fig. 3. (A) Amplification curves of QPCR for synthetic BKV DNA (1 × 100 to 1 × 107 copies/␮L) and negative control, which contain water. Relative fluorescence units are plotted against cycle number (Ct ). No fluorescence signal was generated in the negative control (line). (B) Standard curve from the same experiment as in (A). The threshold cycle is plotted against the log of the starting copy number.

PCR reaction for both JC and BK viruses. The stochastic positivity result of a JCV positive clinical sample is shown in Table 2. This sensitivity was sufficient to detect polyomavirus DNA in: the urine samples (range 30–108 equivalents/␮L of DNA extract corresponded to 3 × 104 to 1011 equivalents/mL of urine sample); in CSF (one sample: 200 equivalents/␮L of DNA extract corresponded to 5 × 104 equivalents/mL of CSF); and in serum (two positive samples: 15 and 20 equivalents/␮L of DNA extract both corresponded to 104 equivalents/mL of serum). None of the 24 samples from healthy blood donors was positive with QPCR. Together with the sequencing results of the seven QPCR positive only samples, this verified the specificity of the QPCR. Table 2 Real-time PCR quantification results of a 10-fold serially diluted of one clinical sample positive for JCV into the stochastic zone (1–10 copies per PCR reaction) Estimated copy number per PCR reaction

Dilution 1 Dilution 2 Dilution 3 Dilution 4 S.Db

100

10

1a

0

130 108 115 137

3 2 16 25

0 2 0 1

0 0 0 0

11.03

0.95

0

13.14

Four different tubes were analysed per dilution. The estimated DNA concentration, in copies per PCR reaction, in relation to the threshold cycle (Ct ) is shown. a At the estimated concentration of one copy per PCR reaction two out four samples were positive, as predicted for the stochastic zone. b Standard deviation.

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4. Discussion Other quantitative real-time PCRs for detection of BKV DNA and JCV DNA have been developed (Biel et al., 2004; Bossolasco et al., 2005; Leung et al., 2002; Leung et al., 2001; Limaye et al., 2001; Priftakis et al., 2003; Whiley et al., 2001). JCV, BKV, and SV40 have been associated with several human diseases. The causal relationships between JCV and progressive multifocal leukoencephalopathy, and between BKV and allograft nephropathy and hemorrhagic cystitis are highly probable. The pathogenicity of SV40 is still uncertain, there is indication that the pathogenic spectra of BKV, JCV, and SV40 may overlap (Li et al., 2002; Randhawa et al., 2001). Co-infection with BKV and SV40 (Li et al., 2002), and with BKV and JCV (Randhawa et al., 2001) in some renal transplant recipients with established PVN has been demonstrated. A sensitive method that carries a low risk of false positivity is needed for the detection of the human polyomaviruses. The intention with the presented method is that a positive result can be typed by sequencing or by reanalysis with methods specific for each of the three viruses. Another use of a broadly amplifying PCR, such as presented here, is for identifying unknown primate polyomaviruses. The conservation of the VP2 region chosen is strong enough to expect a relatively broad range of detection. The identification of a chimpanzee polyoma virus with a broadly targeted PCR (Johne et al., 2005) illustrates that such aims are realistic. The presented method could therefore be useful in screening for unknown polyomaviruses. The principle of broad targeting is to create screening instruments. If the results are positive, additional virus specific PCRs may or may not need to be run. In routine diagnostics, approximately 1% of the samples may be positive (experience of JB). With the broadly targeted test, 99 of 100 samples may be discarded as negative after only one analysis. In order to obtain virus-specific information for BK, JC and SV40 viruses, and for detecting double infections, PCRs specific for the three viruses (i.e. 3 analyses) will need to be run on the sample: in total a maximum of 103 analyses. Compared to running 300 virus-specific reactions this is a considerable saving. For many clinical purposes, it is sufficient to know that a polyomavirus may be involved in the disease, without further virus typing. In this study, a broadly amplifying screening QPCR for the detection of the three polyomaviruses BKV, JCV and SV40 in clinical samples was developed. The high degree of conservation, and the opportunity of avoiding the risk of false positive results due to contamination by common laboratory plasmids containing the TAg gene of SV40 (Lopez-Rios et al., 2004), lead to the selection of degenerate primers from a conserved region of the VP2 gene of JCV, BKV, and SV40. Healthy people can have polyomavirus DNA in peripheral blood (Dolei et al., 2000). It is probable that quantification can distinguish between such apathogenic viral replication from pathogenic. To avoid occasional false-negative results, a routine version of the described method may have to include inhibition controls with known amounts of polyomavirus DNA.

The lowest DNA concentration detected was 10 copies per PCR reaction of SV40 plasmid DNA and 1–10 copies per PCR reaction for JCV, BKV and SV40 synthetic DNA. In contrast to a nested PCR (Bogdanovic et al., 1994), which may take 6 h, and is laborious, the presented QPCR can be accomplished in 2 h. The DNA preparation step takes 1 h and no post-amplification steps are required, if typing is not necessary. The comparative nested PCR method was followed exactly as published (Bogdanovic et al., 1994), i.e. including its sample preparation step. The occasional false negative outcome of the nested PCR was probably caused by the absence of a nucleic acid extraction step; thus, although the nested PCR had a high sensitivity versus naked standard DNA, it could not use the sample DNA as efficiently as the new method. There are arguments for and against this comparison; however, it can be argued that it is the final result that counts. In 41 clinical samples from transplant patients previously tested by nested PCR, 31 were positive with QPCR, whereas 24 were positive by nested PCR. This indicates that QPCR has a high sensitivity. The specificity is probably also high, as sequencing and/or the nested PCR indicated that all QPCR positive samples were truly positive. The positive outcome of QPCR in a CSF sample from a patient with progressive multifocal leukoencephalopathy indicated that this QPCR could be used for diagnosing progressive multifocal leukoencephalopathy patients, although this should be evaluated with more samples. In conclusion, the broadly amplifying QPCR was more sensitive than a nested PCR for the detection of BKV and JCV, and it was sensitive for SV40 DNA detection. As such, broadly amplifying QPCR appears a reliable diagnostic method for screening and quantification of BKV, JCV, and SV40 DNA in clinical samples. The ability to detect all three viruses is clinically useful for diagnosis of CNS disease, which can be caused by several of them. A typing strategy, based on sequencing or strain specific PCRs, will have to be established. The broadly amplifying QPCR may be useful for studies on the relation of polyomaviruses to tumours. Acknowledgments The work was supported by local funds at the Uppsala Academic Hospital and given to Jonas Blomberg. We wish to thank Professor G¨oran Magnusson from the Department of Medical Biochemistry and Microbiology, BMC, Uppsala University, Uppsala, Sweden for provision of the SV40 plasmid. References Arthur, R.R., Shah, K.V., 1999. Polyomaviruses BK and JC. In: Lennette, E.H., Smith, T.F. (Eds.), Laboratory Diagnosis of Virall Infections. Marcel Dekker, New York, pp. 721–730. Arthur, R.R., Shah, K.V., Baust, S.J., Santos, G.W., Saral, R., 1986. Association of BK viruria with hemorrhagic cystitis in recipients of bone marrow transplants. N. Engl. J. Med. 315, 230–234. Biel, S.S., Nitsche, A., Kurth, A., Siegert, W., Ozel, M., Gelderblom, H.R., 2004. Detection of human polyomaviruses in urine from bone marrow transplant patients: comparison of electron microscopy with PCR. Clin. Chem. 50, 306–312.

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