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

Journal of Virological Methods 104 (2002) 59–67

Real-time PCR methods for independent quantitation ofTTV and TLMV

Eva M. Moen, Jowita Sleboda 1, Bjørn Grinde *Department of Virology, National Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway

Received 1 November 2001; received in revised form 20 February 2002; accepted 21 February 2002

Abstract

There is considerable interest in the possible clinical effects of the human circoviruses TT virus (TTV) and TTV-likemini virus (TLMV). Most people appear to have at least one of these viruses replicating actively in their bodies, thusmere correlation of the presence of virus and disease states are probably less informative than a quantitative analysisof viraemia. Real-time PCR based methods, with either SYBR Green or TaqMan probe, designed to quantitateselectively TTV and TLMV are described. The suggested TaqMan-based protocols were suitable for quantitation ofviruses in the range of 102–109 copies/ml of sample; and proved, by sequencing of PCR products, to be specific foreach of the two viruses. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: TTV; TLMV; Real-time PCR; Quantitation; Circovirus

www.elsevier.com/locate/jviromet

1. Introduction

Circoviruses are non-enveloped particles with acircular, single-stranded DNA genome. There aretwo known main human circoviruses, TT virus(TTV) (Nishizawa et al., 1997; Mushahwar et al.,1999) and TTV-like mini virus (TLMV) (Taka-hashi et al., 2000b), with genomes of approxi-mately 3850 and 2860 nucleotides (nt),respectively. TTV was thought originally to bepathogenic for the liver (Okamoto et al., 1998),

however, although a slight increase in circulatingliver enzymes was found in an infected chim-panzee (Tawara et al., 2000), it seems unlikelythat the virus causes severe damage to the humanliver (Kato et al., 2000a; Pistello et al., 2001). Onthe other hand, it is too early to conclude that thevirus is completely benign, a variety of otherpossible pathological consequences of TTV infec-tion has been examined (Christensen et al., 2000;Seemayer et al., 2001; Rodriguez-Inigo et al.,2001).

Recent studies describe TTV prevalences of upto 90% (Huang et al., 2001; Takahashi et al.,2000a); TLMV may be less prevalent, yet it ap-pears to be active in a majority of humans (Nieland Lampe, 2001; Moen et al., 2002). While mostindividuals probably have low titres of TTV, close

* Corresponding author. Tel.: +47-22-042-420; fax: +47-22-042-447.

E-mail address: [email protected] (B. Grinde).1 Present address: Complete Genomics AS, N-0313 Oslo,

Norway.

0166-0934/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

PII: S0 166 -0934 (02 )00039 -3

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E.M. Moen et al. / Journal of Virological Methods 104 (2002) 59–6760

to, or below, the detection limit, some sera con-tain more than 108 copies/ml (Pistello et al., 2001).Thus, in order to investigate a possible pathogenicrole of TTV and TLMV, viral titres are pre-sumably more interesting than the mere presenceof detectable amounts of virus. The level of vi-raemia as an important marker for pathogenesishas been demonstrated in several viral infectionssuch as HIV, hepatitis C virus and cy-tomegalovirus (Berger et al., 1998; Mellors et al.,1996; Spector et al., 1999).

Various methods for the quantitation of TTVhave been described, including serial dilutioncombined with PCR (Christensen et al., 2000),and real-time PCR (Kato et al., 2000b; Pistello etal., 2001); but the recent discovery of TLMV, andthe increased knowledge of TTV sequence hetero-geneity, have left these protocols less than opti-mal. The methods of choice should detect ideallyall strains of TTV and TLMV, and at the sametime distinguish between these two viruses. Fur-thermore, the methods should be sufficiently sen-sitive to yield results even when there are few viralgenomes in the sample. The protocols describedbelow attempts to fulfil these criteria.

2. Materials and methods

2.1. Primers

Comparison of the two human circoviruseswith related animal viruses reveals a distinct con-served region of approximately 150 nt, in TLMVlocated just upstream of the ORF 2, while in TTVlocated within the ORF 2 (Takahashi et al.,2000b). This may be the only segment that allowsthe design of primers that can be expected toamplify most strains; however, as much of thesequence is shared between TTV and TLMV, theprimers should be designed for the purpose oftargeting both viruses, or just one of them. Forthe present purpose, outer primers were designedto amplify both viruses, while the use of TTV orTLMV specific inner primers should allow forspecific detection.

After investigating various primer combina-tions, two sets of primers, one for TTV and one

for TLMV, were found to be the most suitable fortype specific, real-time PCR: TTVf, 5�-GTT TTCTAC GCC CGT CC-3� (115–131); TTVr, 5�-CCTTGA CTC CGG TGT GTA A-3� (210–192);TLMVf, 5�-AGT TTA TGC CGC CAG ACG-3�(193–210); and TLMVr, 5�-CCC TAG ACT TCGGTG GTT TC-3� (287–268). A third primer setwas designed outside, but overlapping, the aboveprimers for the purpose of a two-step PCR: TTV/TLMVf, 5�-TCC GAA TGG CTG AGT TT-3�(102–118); TTV/TLMVr, 5�-CGA ATT GCCCCT TGA CT-3� (219–203); as well as a TaqManprobe to bind between the inner primers: TTV/TLMVpb, (6-FAM)5�-ACT CAC CTH CGGCAC CCG C-3�(TAMRA) (191–173). The latterthree oligonucleotides were designed to serve bothTTV and TLMV. Numbers for the TLMV spe-cific primers are according to the reference strainTLMV CBD231 (GenBank acc. no. AB026930),the others according to the TTV genome TA278(GenBank acc. no. AB008394).

The design of primers and probe was re-evalu-ated in February 2002 after the acceptance of thepaper, in order to take into account sequencesmade available more recently. The evaluation in-cluded 65 entries of TLMV and 289 entries ofTTV. Our recommendation would be to retain theabove primers, but, in order to detect minoritystrains, four of the primers may be mixed with thefollowing slightly modified versions: TTVf, 5�-GTT TTC CAC GCC CGT CC-3�; TTVr, 5�-CCTTGA CT(T/G) CGG TGT GTA A-3�; TTV/TLMVf, 5�-TGA CGA ATG GTA GAG TTT-3�;and TTV/TLMVr, 5�-CGA ATT GGC CCT TAACC-3�. There will still be mismatches, but as the 3�ends of the primers are reasonably conserved, itshould be possible to detect even strains that arenot completely identical.

2.2. PCR protocols

In real-time PCR, the concentration of PCRproduct is measured as the reaction proceeds(Heid et al., 1996). The number of cycles requiredto bring the amount of product above a certainthreshold, the CT value, correlates inversely withthe concentration of template. In the present in-vestigation the Applied Biosystems GeneAmp

E.M. Moen et al. / Journal of Virological Methods 104 (2002) 59–67 61

5700 Sequence Detector was used for the real-timePCR, employing either the TaqMan probe ap-proach or the SYBR Green approach, in proto-cols as recommended by Applied Biosystems.Briefly, the TaqMan probe is an oligonucleotidedesigned to bind to the template between the PCRprimers. It carries a reporter dye at one end and aquencher dye at the other; the Taq DNA poly-merase cuts the probe during target polymerisa-tion, and thus release the reporter from thequencher. SYBR Green is a dye that change itsfluorophoric characteristics upon intercalatingwith DNA, and thereby offers a method for mea-suring the total amount of DNA produced.

DNA was extracted from 200 �l of sera to testfor viraemia by either QIAamp DNA Blood MiniKit supplied with carrier RNA (Qiagen) or HighPure Viral Nucleic Acid Kit (Roche) kits accord-ing to manufacturers recommendations. Bothmethods provided DNA suitable for the variousPCR protocols. The DNA was eluted in 50 �lelution buffer (in the case of the Roche Kit, the‘elution buffer’, being pure water, was supple-mented with 3 �l 10 mM Tris–HCl pH 8.0, 0.1mM EDTA).

The following quantitative PCR strategies wereused in this study: a two-step real-time PCRapproach using TaqMan probe, and a standardreal-time PCR using either TaqMan probe orSYBR Green. In the two-step PCR strategy apreamplification step of 15 cycles was performedprior to the real-time quantitative PCR. Thepreamplification conditions were: 0.4 �M of eachprimer (TTV/TLMV-f and -r), 2.5 mM MgCl2and 0.2 mM dNTP (with dUTP) final concentra-tions in GeneAmp PCR Gold buffer (AppliedBiosystems), 0.5 U AmpliTaq Gold DNA Poly-merase (Applied Biosystems) and 5 �l DNA ex-tract in 25 �l total reaction volume. The DNApolymerase was activated for 10 min at 95 °C,followed by 15 cycles of amplification: 15 s at95 °C, 15 s at 62 °C and 30 s at 72 °C; and afinal elongation step of 2 min at 72 °C. Thereal-time TaqMan PCR protocol was as follows:0.3 �M of each primer (TTV-f and -r or TLMV-fand -r), 5 mM MgCl2, 0.2 mM dNTP (withdUTP) and 0.2 �M TaqMan probe (TTV/TLMVpb) in TaqMan buffer (Applied Biosys-

tems), 1 U AmpliTaq Gold DNA Polymerase and1.5 �l preamplification product (two-step) or 5 �lDNA extract (standard) in 25 �l total reactionvolume. The DNA polymerase was activated for10 min at 95 °C followed by 40 cycles of amplifi-cation: 15 s at 95 °C and 1 min at 62 °C. In thecase where SYBR Green was used, the real-timePCR conditions were: 0.3 �M of each primer, 2mM MgCl2 and 0.2 mM dNTP final concentra-tions in SYBR Green PCR buffer (AppliedBiosystems), 1 U AmpliTaq Gold DNA Poly-merase in 25 �l total reaction volume, and cyclingconditions as described for the TaqMan protocolabove. The preamplification was optimised as toMg2+ concentration and annealing temperature,the real-time PCRs were optimised as to primerconcentrations.

2.3. Sequencing

TTV or TLMV real-time PCR products, ob-tained by the two-step PCR, were sequenced inboth directions with ABI BigDye Terminator Cy-cle Sequencing Kit (Applied Biosystems) and theprimers of the two real-time PCRs, and analysedon an ABI PRISM 310 Genetic Analyser (AppliedBiosystems) as previously described (Huang et al.,2001). The sequences were edited using Sequencer3.1 (Gene Codes). The sequences were submittedto the GenBank under accession no. AJ315985–AJ316004.

3. Results

3.1. Linearity and specificity of assay

In order to create a standard that could be usedto convert the real-time PCR results to an esti-mate of actual copy number, PCR products cov-ering the region or either TTV or TLMV werequantified by gel electrophoresis along with DNAmass ladders of known concentration. In Fig. 1 isshown the results of testing 10-fold dilutions ofthese standards with the TaqMan based proto-cols. As can be seen, the protocols gave reason-ably linear results when plotting the CT valuesagainst the log of template DNA copies, in either


the range 5–5×107 copies/tube (in the case of asingle PCR protocol, �/�), or the range 0.5–5×105 (in the case of the two-step TTV protocol,�). In the above protocols one copy/sample vol-ume analysed corresponds to 50 copies/ml ofserum.

Standard regression analyses of the linear partsof the curves (using the exponential values of thenumber of DNA copies i.e. 104=4) gave slopecoefficients of: −3.33 for single-step TTV PCR,−3.45 for single-step TLMV PCR, and −2.94for two-step TTV PCR). In a 100% effective PCR(i.e. the DNA doubles in each cycle), the slopeshould be −1/log 2= −3.32, thus the real-timePCR of the purified PCR product appeared to beclose to 100% efficient. The slightly lower effi-ciency observed in the two-step protocol wasprobably due to a less than 100% efficiency overthe 15 cycles of the initial PCR.

In order to verify that the inner primers were

specific for the viruses they were designed for,DNA from ten patients harbouring both TTVand TLMV were first amplified by the generalouter primers, and subsequently by the specificinner primers. The 20 PCR inner products weresequenced, and the derived sequences were usedto probe the GenBank database: the 10 productsobtained by TTV primers clustered with otherTTV sequences, while the 10 products obtained byTLMV primers clustered with TLMV sequences.The sequencing also confirmed that the designedTaqMan probe matched perfectly all the virusesexamined (data not shown).

A total of 33 serial serum samples from 5different individuals were tested for TTV withboth SYBR Green and TaqMan probe protocols.The estimates based on SYBR Green were on theaverage higher than those based on TaqManprobe (6.7 log and 5.9 log virus/ml, respectively).Both methods captured the variations within each

Fig. 1. Standard curves for real-time PCR based on TaqMan probe. TTV (�, �) or TLMV (�) PCR products that encompassedthe various primer sites were quantified by comparison with DNA of known concentration on 2% agarose gels containing ethidiumbromide and were used as templates for generation of real-time PCR standard curves. Two separate dilution series of each standardtemplate were made (DNA copies in sample), and each dilution was tested in duplicate with the protocols described for quantitation.The symbols represent the average CT values of either single PCR protocols (open symbols), or a two-step protocol where thedilutions of the TTV standard template were initially amplified for 15 cycles with primers outside those used for subsequentquantitation (closed circles).


Fig. 2. Comparison of SYBR Green and TaqMan based methods. TTV was quantified by real-time PCR in serial blood samples byeither SYBR Green (�) or TaqMan (�) based protocols. In three of five individuals the two methods gave comparable results(exemplified in panel A), while in two individuals the SYBR Green method gave consistently higher estimates (exemplified in panelB).

individual, the elevated average value for theSYBR Green method was a consequence of con-sistently higher estimates in two of the five sub-jects (Fig. 2).

3.2. Consistency of assay

In order to evaluate the consistency of the TTVTaqMan protocol, triplicate extractions weremade from 3 different samples and each of the 9extracts were analysed in 3 parallels on 3 differentdays. On one of the days, the samples were alsotested by first subjecting them to preamplificationwith the outer primers.

Of the total of 108 real-time PCRs included inthe above exercise, one of the values was totallyoff score: a CT value of 18 where all other resultsfrom the sample in question was above 30. Inthree other samples, one of the CT values weretwo to three units above the other two parallels,an error possibly due to the presence of bubblesor condensation in the tube obscuring the photo-metric registration. A similar frequency of oddvalues was observed in other experiments, and itwas considered reasonable to remove such resultsfrom the data set. When looking at the remainingnumbers, the parallels were, with few exceptions,within one CT unit.

The variances presented in Table 1 were calcu-lated in order to indicate the additional contribu-tion of extraction and day to day differences,compared to the basal variations observed withinexperiments. Variances were higher in the case ofa sample carrying very few viral copies (sampleA), compared to the samples with medium copynumbers (samples B and C). As expected, thevariances based on data from different extracts(column 5) or different days (column 6) werehigher than the within experiment variances(column 4); yet the major contribution to varianceappeared to be due to within experiment parallels(with the possible exception of the low copy num-ber sample A).

The preamplification step did not increase thevariance (column 7). In fact, in the case of the lowcopy number sample the variance actually de-creased following the independent preamplifica-tion of each parallel.

4. Discussion

TTV and TLMV are two distinct viruses, bothwith considerable sequence heterogeneity, somesubtypes within the TTV group have overall nu-cleotide sequence identity as low as 50% (Hallett


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et al., 2000; Tanaka et al., 2001; Biagini et al.,2001), yet there are certain conserved regions thatcan cause primers designed for one virus to am-plify the other (Takahashi et al., 2000b). It ispresumably important to differentiate betweenTTV and TLMV, but at the same time the ex-treme sequence heterogeneity requires primersthat bind to highly conserved regions. The presentset of primers was designed with these constraintsin mind. Besides being useful for real-time PCR,they have been employed successfully in sensitivenested PCR detection protocols, in which 90%(Huang et al., 2001) and 48% (Moen et al., 2002)of a set of Norwegian blood donors were found tobe positive for TTV and TLMV, respectively.

The available sequence data is expanding con-stantly. The primers were reconsidered in Febru-ary 2002 in order to adapt them to strainsdescribed recently. As described in Section 2,three of the primers might be supplemented withone additional version each. Even with these sup-plements there may, however, be occasionalstrains that remain undetected.

It is considered that the present protocols areuseful for quantitation of TTV and TLMV, andthat they that can be used to search for correla-tion with various clinical conditions. Each experi-mental set up should obviously include bothpositive and negative controls. The positive con-trol should preferably be of a fixed concentrationin order to evaluate whether the expected CT

value is obtained. The use of in-tube internalstandards added to each serum was considered,but deemed unnecessary; presumably clinicallyrelevant variations in viraemia are in the order ofone log or more, a variation that should be easyto detect by the present methods without the extracalibration provided by internal standards.

It should be noted that in the case of DNAextracted from clinical material, the efficacy of thePCR may be less than 100%, due to possibleinhibitors of the PCR; in which case the use of thestandard curve will lead to an underestimate ofthe number of viral copies present in the sample.

Although the primers are expected to work wellwith most sequences known at present, somestrains may contain disrupting mutations. TheSYBR Green approach was tested as a way to

overcome partly the problem of sequence hetero-geneity: by avoiding the TaqMan probe the ques-tion of sequence compatibility would be less of anissue, particularly as while mutations in theprimer sequences should only hamper the firstcycle(s), point mutations in the probe region mayalter the yield of signal throughout the PCR.Particularly in the case of viral studies, wheresequence heterogeneity tends to be extreme com-pared to most applications, SYBR Green mightserve a purpose. The disadvantage of SYBRGreen is that measurements may be obscured bythe amplification of non-target sequences andprimer dimers. However, whether this is a prob-lem in a given reaction can be investigated bymelting point (Tm) analysis of the final products:the correct product is expected to have a particu-lar Tm, easily distinguished from primer-dimerartefacts.

For the present purpose, the TaqMan approachwas preferred for the following reasons: the Tm

calculated with SYBR Green varied from sampleto sample; and occasionally the melting pointanalysis gave two peaks even when the productappeared to be homogenous both by elec-trophoresis and sequencing, an observation thatmay have a rational explanation but was stilldiscomforting. In addition, the TaqMan approachgave, on the average, slightly less deviation be-tween parallel samples (data not shown). Whenthe same samples were analysed by both methods,the SYBR Green method did yield consistentlyhigher estimates in the case of samples comingfrom two individuals, while in three other casesthe results were comparable (as exemplified inFig. 2). It is not possible to tell which of the twomethods offered the best estimates in the formersamples, the different estimates were not due tomismatches to the TaqMan probe.

As shown in Fig. 1, it is feasible to enhance thesensitivity of the quantitation by adding an outerPCR prior to the real-time PCR. If sufficientamount of virus is present, the single-step proce-dure has the following advantages: the two-stepapproach requires the sequence match of at leastone more primer, and it exposes the proceduresfor the problems of contamination. (Real-timePCR with a single primer set does not require any


opening of tubes containing PCR products,which reduces the contamination problem signifi-cantly.) Furthermore, the present outer primersamplify both TTV and TLMV, thus if a samplecontains much more TTV than TLMV, the two-step procedure is not recommended for TLMVquantitation as the production of a large amountof TTV may inhibit either the first or the secondamplification of TLMV (or vice versa).

The choice of universal outer primers wasbased on two factors: one, the observation thatit seemed difficult to design outer primers spe-cific for either TTV or TLMV that could beexpected to deal with the known sequence varia-tion within each group; and two, that the use ofa common set of outer primers reduced the num-ber of PCRs required for the analysis of bothviruses.

As to the problem of contamination, it is pos-sible to include the additional measure of con-tamination prevention of the uracil DNAglycosidase (UNG) system (Pang et al., 1992). Inthe present amplification protocols dUTP wasincluded, which facilitates the destruction ofDNA by UNG, but the enzyme itself was notincluded. The rational for this choice was thatthe enzyme might reduce the yield of PCR prod-ucts and thus the sensitivity of the system, andthat it could be added in case a problem ofcontamination should arise.

It is useful to have data that suggest howmany parallels are required, and how the paral-lels should be prepared, in order to obtain asatisfactory estimate of viral titre. According tothe present variance analysis (Table 1), the majorcomponent of variance is within the parallels ofa single extract analysed at the same time. Thuspreparing additional extracts, or undertaking theanalysis on separate days, should, for most pur-poses, be unnecessary. Moreover, where a rea-sonable amount of virus is present, threeparallels are sufficient to generate a reasonablyaccurate estimate. Three parallels, however, is anabsolute minimum in order to single out the oc-casional incongruent result; furthermore, in thecase of samples with few viral particles present,more parallels may be advantageous.

Acknowledgements

We would like to thank Tom Øystein Jonassenfor design of primers and help with statisticalanalysis, and Sanela Numanovic for technicalassistance.

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Documents

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