Real-time PCR methods for independent quantitation of TTV and TLMV

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Abstract

There is considerable interest in the possible clinical effects of the human circoviruses TT virus (TTV) and TTV-like mini virus (TLMV). Most people appear to have at least one of these viruses replicating actively in their bodies, thus mere correlation of the presence of virus and disease states are probably less informative than a quantitative analysis of viraemia. Real-time PCR based methods, with either SYBR Green or TaqMan probe, designed to quantitate selectively TTV and TLMV are described. The suggested TaqMan-based protocols were suitable for quantitation of viruses in the range of 102–109 copies/ml of sample; and proved, by sequencing of PCR products, to be specific for each of the two viruses.

Introduction

Circoviruses are non-enveloped particles with a circular, single-stranded DNA genome. There are two known main human circoviruses, TT virus (TTV) (Nishizawa et al., 1997, Mushahwar et al., 1999) and TTV-like mini virus (TLMV) (Takahashi et al., 2000b), with genomes of approximately 3850 and 2860 nucleotides (nt), respectively. TTV was thought originally to be pathogenic for the liver (Okamoto et al., 1998), however, although a slight increase in circulating liver enzymes was found in an infected chimpanzee (Tawara et al., 2000), it seems unlikely that the virus causes severe damage to the human liver (Kato et al., 2000a, Pistello et al., 2001). On the other hand, it is too early to conclude that the virus is completely benign, a variety of other possible pathological consequences of TTV infection has been examined (Christensen et al., 2000, Seemayer et al., 2001, Rodriguez-Inigo et al., 2001).

Recent studies describe TTV prevalences of up to 90% (Huang et al., 2001, Takahashi et al., 2000a); TLMV may be less prevalent, yet it appears to be active in a majority of humans (Niel and Lampe, 2001, Moen et al., 2002). While most individuals probably have low titres of TTV, close to, or below, the detection limit, some sera contain more than 108 copies/ml (Pistello et al., 2001). Thus, in order to investigate a possible pathogenic role of TTV and TLMV, viral titres are presumably more interesting than the mere presence of detectable amounts of virus. The level of viraemia as an important marker for pathogenesis has been demonstrated in several viral infections such as HIV, hepatitis C virus and cytomegalovirus (Berger et al., 1998, Mellors et al., 1996, Spector et al., 1999).

Various methods for the quantitation of TTV have been described, including serial dilution combined with PCR (Christensen et al., 2000), and real-time PCR (Kato et al., 2000b, Pistello et al., 2001); but the recent discovery of TLMV, and the increased knowledge of TTV sequence heterogeneity, have left these protocols less than optimal. The methods of choice should detect ideally all strains of TTV and TLMV, and at the same time distinguish between these two viruses. Furthermore, the methods should be sufficiently sensitive to yield results even when there are few viral genomes in the sample. The protocols described below attempts to fulfil these criteria.

Section snippets

Primers

Comparison of the two human circoviruses with related animal viruses reveals a distinct conserved region of approximately 150 nt, in TLMV located just upstream of the ORF 2, while in TTV located within the ORF 2 (Takahashi et al., 2000b). This may be the only segment that allows the design of primers that can be expected to amplify most strains; however, as much of the sequence is shared between TTV and TLMV, the primers should be designed for the purpose of targeting both viruses, or just one

Linearity and specificity of assay

In order to create a standard that could be used to convert the real-time PCR results to an estimate of actual copy number, PCR products covering the region or either TTV or TLMV were quantified by gel electrophoresis along with DNA mass ladders of known concentration. In Fig. 1 is shown the results of testing 10-fold dilutions of these standards with the TaqMan based protocols. As can be seen, the protocols gave reasonably linear results when plotting the CT values against the log of template

Discussion

TTV and TLMV are two distinct viruses, both with considerable sequence heterogeneity, some subtypes within the TTV group have overall nucleotide sequence identity as low as 50% (Hallett et al., 2000, Tanaka et al., 2001, Biagini et al., 2001), yet there are certain conserved regions that can cause primers designed for one virus to amplify the other (Takahashi et al., 2000b). It is presumably important to differentiate between TTV and TLMV, but at the same time the extreme sequence heterogeneity

Acknowledgements

We would like to thank Tom Øystein Jonassen for design of primers and help with statistical analysis, and Sanela Numanović for technical assistance.

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    Present address: Complete Genomics AS, N-0313 Oslo, Norway.

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