Journal of Virological Methods 132 (2006) 127–134

Absolute quantitation of Marek’s disease virus and Herpesvirus of turkeysin chicken lymphocyte, feather tip and dust samples using real-time PCR

Aminul Islama,∗, Brian F. Cheethamb, Timothy J. Mahonyc,Peter L. Youngc, Stephen W. Walkden-Browna

a Centre for Animal Health and Welfare, School of Rural Science and Agriculture, University of New England, Armidale, NSW 2351, Australiab Molecular and Cellular Biology, School of Biological, Biomedical and Molecular Sciences, University of New England, Armidale, NSW 2351, Australia

c Department of Primary Industries and Fisheries, Queensland Biosciences Precinct, St. Lucia, Qld 4072, Australia

Received 19 July 2005; received in revised form 11 September 2005; accepted 3 October 2005Available online 14 November 2005

Abstract

The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek’s disease virus serotype 1(MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNAc were foundt ss than 5%f tips frome d curves, ands ntification.T f samples.©

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opies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assayso be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being leor Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and featherxperimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standartandard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quahese methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range o2005 Elsevier B.V. All rights reserved.

eywords: Real-time PCR; qPCR; Viral quantitation; Marek’s disease virus serotype 1; Herpesvirus of turkeys

. Introduction

Marek’s disease virus serotype 1 (MDV1) and herpesvirusf turkeys (HVT) are DNA viruses belonging to the familyerpesviridae, sub-familyAlphaherpesvirinae and genusMar-ivirus (Van Resenmortel et al., 1999; Buchen-Osmond, 2004).DV1 is oncogenic and is the etiological agent of Marek’s dis-ase (MD) of poultry (Churchill and Biggs, 1967; Nazeriant al., 1968; Solomon et al., 1968). Clinical signs of MD

nclude immunosuppression, polyneuritis and T-cell lymphomaormation in the visceral and ectoderm-derived tissues (Witternd Schat, 2003). MD is well controlled by vaccination withononcogenic HVT, MDV serotype 2 (MDV2) or attenuatedDV1 vaccine viruses (Witter, 2001). Both vaccinal and viru-

ent viruses can coexist in the same host (Witter and Lee, 1984;itter et al., 1987; Lee et al., 1999; Islam et al., 2005a) and

∗ Corresponding author. Tel.: +61 2 6773 2670; fax: +61 2 6773 3922.E-mail address: aislam@une.edu.au (A. Islam).

their replication cycle is typical of other cell-associated heviruses (Biggs, 2001). Fully productive MDV replication occuin the feather follicle epithelium (FFE) and cell-free virus is sin skin and feather debris (Calnek et al., 1970; Carrozza et1973) which contaminates the poultry environment and cotutes the source of further infection through inhalation ofparticles.

For molecular diagnosis of MDV infection using diffeent tissues, serotype-specific standard polymerase chaintion (PCR) assays have been extensively used during thedecade (Becker et al., 1993; Davidson et al., 1995; Youand Gravel, 1996; Burgess and Davison, 1999). These methodare mostly qualitative (Davidson et al., 1995; Handberg et2001; Walkden-Brown et al., 2003) or at best semi-quantitativ(Bumstead et al., 1997; Burgess and Davison, 1999; Redal., 2000) and require labour-intensive post-PCR handlingend point analysis of the PCR product.

Recently, quantitative real-time PCR (qPCR) assaysemerged as the method of choice for quantitation of viral nuacids (Niesters, 2001; Mackay et al., 2002). This method is rapid

166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2005.10.009

128 A. Islam et al. / Journal of Virological Methods 132 (2006) 127–134

sensitive and reproducible with a wide dynamic range. Otherbenefits include a minimal risk of carry-over contamination dueto the absence of post-PCR manipulation of the amplicon. Real-time PCR technology has recently been used for the quantitationof MDV genomes mostly confined to quantitation of MDV1(Kaiser et al., 2003; Levy et al., 2003; Baigent et al., 2005).We have previously developed qPCR assays for all three MDVserotypes, allowing relative quantitation of viral DNA using sep-arate or duplex assays (Islam et al., 2004). In these assays, thestandard curve comprised a serial dilution of DNA from infectedspleen tissue and viral content was expressed as a ratio of viralto host DNA content (normalized value) or as a ratio of thenormalized value of the sample to a calibrator sample (relativeabundance) (Livak, 1997). However, these assays did not allowquantitation of viral genome copy number. An absolute quantita-tion method to determine MDV genome copy number providesmore meaningful data than the relative quantitation method forrelating infection and disease (Baigent et al., 2005).

This paper describes the development and validation of plas-mid standards for the previously reported MDV1 and HVT qPCRassays. It also describes the conversion of our previous stan-dards derived from the spleen of infected chickens to absolutestandards, based on the demonstration of parallelism with theplasmid standard curve and the determination of viral copynumber for each standard. Both methods were applied to thequantitation of both viruses in PBL, feather tip and dust sam-p tatioa theh d foe

2

2

peds inq

2.1.1. Development of constructsA 196 bp fragment of themeq gene (MDV1 specific) and

a 350 bp fragment ofsorf1 (HVT specific) gene were ampli-fied by standard PCR using primers shown inTable 1. The25�l amplification reactions contained 1�mol of each oligonu-cleotide primer, 1.8 mM MgCl2, 0.2 mM dNTPs, 1× reactionbuffer (Fisher Biotec, Perth, Australia), 1 unit ofTaq DNApolymerase and approx 1 ng of template DNA. The reactionmixtures were amplified for thirty cycles (90 s at 94◦C, 60 s at60◦C and 120 s at 72◦C) in a Corbett FTS-320 thermal cycler(Corbett Research, Australia). The PCR products were purifiedby the WizardR DNA purification system (Promega, Madison,USA) and then ligated into the T-tagged site of pGEM-T easyvector, according to the manufacturer’s instructions (Promega).After 1 h of incubation, the ligation mix was transformed intocompetentE. coli (DH5�) and positive transformants were iden-tified by blue/white screening. The size of the cloned ampliconswas confirmed by the analysis of plasmid DNA extracted fromthe transformedE. coli (DH5�) usingEcoRI digestion and gelelectrophoresis, and the sequence of the inserts was determinedby Newcastle DNA, University of Newcastle, Australia. ThepGEM-T easy vector containing the fragment ofmeq gene wasnamed pAI-meq and that containing the fragment ofSorf1 geneas pAI-sorf1.

2ml

ci rer’si midD ts oft stan-d um-b eightoop tectt NAs

TP s use

T nce (bp)

qTTAGGACCC

CGGCACCGC

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TTGCGTC

les. The development of these assays will enable quantind differentiation of coexisting MDV1 and HVT viruses inost and their environment for both research purposes anffective monitoring of MD status in the poultry industry.

. Materials and methods

.1. Development of plasmid standard

MDV1- and HVT-specific plasmid standards were develoeparately using part of themeq and sorf1 genes that contaPCR primers described byIslam et al. (2004).

able 1rimers and probes used in quantitative real-time PCR assay and primer

arget gene Primer/probe seque

PCR assaymeq (MDV1) (Islam et al., 2004) Probe: 5′-(FAM)CGTC

F primer: 5′-GGAGCCR primer: 5′-ATCTGG

sorf1 (HVT) (Islam et al., 2004) Probe: 5′-(CY5) AACCF primer: 5′-GGCAGAR primer: 5′-TGTCCA

tandard PCR assaymeq (MDV1) F primer: 5′-GAATCT

R primer: 5′-ATCTGG

sorf1 (HVT) F primer: 5′-AAGCGCR primer: 5′-TATGGA

n

r

.1.2. Preparation of plasmid DNA for standard curvesPlasmid DNA was extracted and purified from a 500

ulture of transfectedE. coli using the WizardR plus Max-preps DNA purification system, according to the manufactunstructions (Promega). The concentration of purified plasNA was determined using the mean of three to four aliquo

wo-fold dilutions by agarose gel electrophoresis againstards containing known amounts of DNA. Plasmid copy ners were then determined by calculating the molecular wf each cloned plasmid. Master stock containing 5× 105 copiesf pAI-meq plasmid and 7.5× 105 copies of pAI-sorf1 plasmider 5�l were used to prepare a 10-fold dilution series to de

he lower limit of detection of each assay. Purified plasmid Dtandards were stored at−20◦C.

d in amplification of fragments ofmeq andsorf1 genes by standard PCR for cloning

Amplicon

CCGAGGATCCCGAACAGG (BHQ-1)-3′ 67GAGGCTTTATG-3′GAATACAAGGAA-3′

GCTTGTGGACGTCTTC(BHQ-3)-3′ 76CGCGTTGTAT-3′TCGAGACTATCC-3′

TGCATTGTGTC-3′ 196GAATACAAGGAA-3′

TATGTGTAGG-3′ 350ATGCAGTTGG-3′

A. Islam et al. / Journal of Virological Methods 132 (2006) 127–134 129

2.2. Quantitative real-time PCR (qPCR)

2.2.1. Primer/probe setsThe oligonucleotide sequences of MDV1 and HVT spe-

cific primers and the TaqMan fluorogenic probes are shown inTable 1.

2.2.2. qPCR protocolThe qPCR parameters followed were those outlined in the

Rotor Gene 3000 Real-Time Amplification Operator’s Man-ual v 4.6 (Corbett Research, Sydney, Australia) and previouslydescribed byIslam et al. (2004). Briefly, each reaction contained0.3�M of each primer and 0.2�M of the corresponding probe,12.5�l of Platinum® Quantitative PCR System-UDG (Invitro-gen Australia Pty Limited), 5�l of DNA template (25 ng) in atotal reaction volume of 25�l. Duplicate reactions were run foreach sample.

The cycling parameters consisted of: 50◦C for 2 min, 95◦Cfor 2 min, followed by 40–45 cycles consisting of denaturation at94◦C for 15 s and annealing/extension at 60◦C for 45 s. Ampli-fication and data acquisition were carried out using Rotor Gene3000 real-time PCR machine (Corbett Research, Sydney).

Results of qPCR assays were analyzed using Rotor-GeneAnalysis software version 4.6 supplied with the Rotor Genemachine (Corbett Research). The default settings of the pro-g selinf r sew umbo

2as

d mids d inS thel hes Austd aysw cieno ford ardi sayC latec runa meao ssayr aredr

2om

ie int h thp

2.3. MD viruses

Experimental samples in this study came from chickensinfected with the MPF57 strain of MDV1 and the FC 126 strainof HVT. Field dust samples contained MDV1 of unknown origin.

2.4. Samples for measurement of MDV1 and HVT load

Spleen samples were from a previously reported isolatorexperiment using female Cobb broiler chickens (Islam et al.,2001). PBLs were obtained from HVT-vaccinated female Cobbbroiler chickens vaccinated at hatch (day 0) with 8000 pfu ofHVT and challenged at day 5 with 50 pfu of MDV1 (strainMPF57), maintained in isolators and sampled weekly over a 5-week period (Islam et al., 2005b). Wing feathers were collectedfrom MDV1- and HVT-infected female Cobb broiler chickensin a separate isolator experiment (Islam, A.F.M.F., Walkden-Brown, S.W., Underwood, G.J., Groves, P.J., unpublished). Thebirds were vaccinated with HVT vaccine (8000 pfu) at day 0and challenged with the MPF57 strain of MDV1 (50 pfu) at day5. Feather samples were collected at day 56 post-vaccination.A representative poultry dust sample from an HVT-vaccinatedcommercial broiler chicken farm was also included. Dust wassimply scraped from available surfaces within the shed on day35 after placement.

MDV1 and HVT viral loads in spleen, PBL and feather tipsw hostc oidc ndh VCNp

2

fol-l tips( ingtt du TM3a

2

ed ino asterm em-p medi atory,e andg rosol-b andf l andp lu-t o,C

ram were used to define both the threshold value and baor analysis of the raw data. A standard curve for each primeas generated in each assay and used to derive the copy nf target sequences in unknown samples.

.2.3. Validation of assaysThe sensitivity of the assays for MDV1 and HVT w

etermined by running 10-fold serial dilutions of the plastandards with known numbers of copies, as describeection2.1.2. The lower limit of detection was defined as

owest dilution in the 10-fold series that amplified reliably. Tpecificity of the primers was previously assessed usingralian field and vaccine strains of MDV (Islam et al., 2004). Toetermine the reproducibility of MDV1 and HVT qPCR assith the new plasmid standards the intra-assay coeffif variation (CV) was calculated by taking the mean CVuplicate Ct and calculated copy number for plasmid stand

n all runs of the same assay (MDV1 or HVT). The inter-asV was determined by comparing the mean Ct and calcuopy number for each standard in three different assaynd determining the CV for each across assays. Thef these CVs formed the inter-assay CV. The different auns were performed on different days using freshly prepeagents.

.2.4. Conversion of previous MDV1 and HVT standardsThe previous MDV1 and HVT standards extracted fr

nfected spleen tissue and used for relative quantitation (Islamt al., 2004) were quantified in terms of viral copy number

hree separate assays after confirmation of parallelism witlasmid standard curve.

eter

-

t

s

dsn

e

ere expressed as viral copy number (VCN) per millionells (MHC) assuming a DNA yield of 2.5 pg per avian diplell (Gregory, 2005) and equal extraction efficiency of viral aost DNA. Viral load in dust samples was expressed aser mg of dust.

.5. Template extraction and quantification

DNA samples were extracted from spleen (10 mg), PBLowing Ficoll-Paque separation from 1–1.5 ml blood, feather4–6) and dust (5 mg), using a QIAamp DNA mini kit accordo the manufacturer’s instructions (Qiagen, Clifton Hill , Aus-ralia) and stored at−20◦C. Extracted DNA was quantifiesing spectrophotometric analysis (BIO-RAD, SmartSpec000) and diluted to a fixed concentration of 5 ng/�l before uses template.

.6. Contamination control

The plasmid DNA standards were produced and preparne laboratory. The preparation of qPCR reagents and the mixture, the extraction of the DNA and the addition of the tlate to the PCR mixture and the thermal cycling were perfor

n three different, well-separated rooms in a separate laborach room having its own dedicated set of micropipettesowns. Other precautions included systematic use of aearrier-protected pipette tips, frequent changes of gloves

requent decontamination of working surfaces with ethanoeriodic cleaning of other equipment with DNA cleaning so

ion (DNA AwayR, Molecular Bioproducts, Inc., San DiegA).

130 A. Islam et al. / Journal of Virological Methods 132 (2006) 127–134

3. Results

3.1. Plasmid constructs

A 196 bp fragment of the MDV1-specificmeq gene wasamplified by PCR using primers (Table 1) that flank the regionamplified in the previously reported qPCR assay for MDV1(Islam et al., 2004). The 196 bp fragment was cloned into theplasmid pGEM-T easy (Promega Corporation) to generate theplasmid pAI-meq. DNA sequencing was used to show that thisplasmid contained the correct region of themeq gene (data notshown). Similarly, a 350 bp fragment of the HVT-specific genesorf1 (Table 1) was amplified and cloned into pGEM-T easy togenerate the plasmid pAI-sorf1.

3.2. Sensitivity

Figs.1A and2A show the amplification plots ofmeq (MDV1)and sorf11 (HVT) genes for 10-fold dilution series of the

Fdr(lcs

pAI-meq and pAI-sorf1 plasmid standards, respectively. Thelog–linear standard curves generated from these plots were avery good fit (R2 > 0.99; Figs.1B and 2B) demonstrating theaccuracy of amplification over a wide range of concentrations of5–5× 104 copies for pAI-meq plasmid and 75–7.5× 105 copiesfor pAI-sorf1 plasmid standards. The detection limit, defined asthe lowest dilution in the 10-fold series which amplified reliably,was 5 copies for the MDV1 assay and 75 copies for the HVTassay.

3.3. Reproducibility

Standard curves for each assay were highly reproducible withno significant difference in slopes between different runs of thesame assay (P < 0.05, Figs.1C and2C). The intra- and inter-assay variations are shown inTable 2. For the MDV1 and HVTassays Ct values had a mean intra-assay CV below 1% and amean inter-assay CV below 4%. Calculated plasmid copy num-ber had mean intra- and inter-assay CVs of below 20%.

ig. 1. MDV1 assay. (A) Amplification plot ofmeq gene showing a 10-foldilution series ofmeq-plasmid copies (5–50,000, from right to left). The releasedeporter fluorophore is plotted as a function of the amplification cycle number.B) Standard curve [y = 40.4948− 3.3171 logx, with R2 = 0.995]. The dottedines represent the 95% confidence limits of the assay. (C) Plots of three standaurves generated in separate assays. The slopes and intercepts did not difignificantly.

F1TcTtn

rdfer

ig. 2. HVT assay. (A) Amplification plot ofsorf1 gene of HVT showing a0-fold dilution series ofsorf1-plasmid copies (75–750,000, from right to left).he released reporter fluorophore is plotted as a function of the amplificationycle number. (B) Standard curve [y = 42.3020− 3.266 logx, with R2 = 0.995].he dotted lines represent the 95% confidence limits of the assay. (C) Plots of

hree standard curves generated in separate assays. The slopes and intercepts didot differ significantly.

A. Islam et al. / Journal of Virological Methods 132 (2006) 127–134 131

Table 2Overall mean intra- and inter-assay coefficient of variation (CV, percentage)based on Ct values and calculated plasmid copy numbers (CPCN) of standards

Assays Mean intra-assay CV Mean inter-assay CV

Ct (%) CPCN (%) Ct (%) CPCN (%)

MDV1 0.7 17.8 3.1 19.4HVT 0.7 15.7 1.9 15.3

3.4. Conversion of previous MDV1 and HVT standards

Figs.3A and4A show parallelism of standard curves gener-ated from MDV1- and HVT-positive reference standards andpAI-meq and pAI-sorf1 plasmid standards. Figs.3B and 4Bshow linear regression plots of pAI-meq and pAI-sorf1 plas-mid copy number against the DNA concentrations in the oldMDV1 and HVT standard curves respectively. These data enableabsolute quantification of the old standards and conversion ofprevious assays from relative quantification, to absolute quan-tification.

3.5. Absolute quantitation of MDV1 and HVT genome inPBL, feather tip and dust samples

MDV1 and HVT genome copy numbers in infected feathertips and field dust samples at particular time points are shownin Figs. 5 and 6, respectively. In feather tips, the MDV1 load invaccinated and unvaccinated chickens was similar (107.7–108.1

copies/million cells) and about 3 logs higher than the HVT load

F DV1p ints ferenceM

Fig. 4. HVT assay. (A) Parallelism of standard curves generated from HVTpositive reference standard and pAI-sorf1 plasmid standard. Each data pointshows mean of three separate assays. (B) Linear regression plot of referenceHVT DNA concentration and pAI-sorf1 plasmid copy number per reaction.

Fig. 5. Mean (±S.E.M.) MDV1 and HVT viral loads in feather tips of Cobbbroiler chickens at day 56 post challenge with MDV1 (MPF57). Chickens werevaccinated with HVT (FT126) at hatch and challenged with MDV1 at day 5.DNA samples extracted from 4–6 feather tips as mentioned in the text. MDV1loads in unvaccinated and vaccinated chickens represent mean of 3 and 12 sam-ples, respectively, and HVT loads represent the mean of 2 samples.

Fig. 6. MDV1 and HVT genome copy number in field poultry dust. A represen-

ig. 3. MDV1 assay. (A) Parallelism of standard curves generated from Mositive reference standard and pAI-meq plasmid standard. Each data pohows mean of three separate assays. (B) Linear regression plot of re DV1 DNA concentration and pAI-meq plasmid copy number per reaction.

tative sample collected at day 35 from an Australian commercial Cobb broilerfarm vaccinated with HVT vaccine.

132 A. Islam et al. / Journal of Virological Methods 132 (2006) 127–134

Fig. 7. Mean (±S.E.M.) MDV1 and HVT viral load in PBL of Cobb broilerchickens at days 5–35 post challenge with MDV1. Chicks were vaccinatedwith HVT (FC126) at hatch and challenged with MDV1 (MPF57) at day 5, soHVT data needs to be interpreted accordingly (i.e. days post vaccination = dayspost challenge + 5). Each data point represents the mean of three chickens (notrepeated measures).

(105.3 copies/million cells) at day 56. However, in field dust theHVT load (105.5 copies/mg of dust) was about 1 log higher thanMDV1 load (104.7 copies/mg of dust) at day 35.

Fig. 7shows the viral load and performance of both virusesin infected PBL samples over time. Mean MDV1 load wasinitially very low (101.7copies/million cells) at 5 dpc andsharply increased (104.2copies/million cells) at 14 dpc with fur-ther increase to 104.9–105 copies/million cells at days 28–35pc respectively. However, there was little variation in HVTload between days 10 and 40 post vaccination (104.2–104.6

copies/million cells).

4. Discussion

This paper describes the development and validation of twoplasmid constructs, pAI-meq (MDV1 specific) and pAI-sorf1(HVT specific), for the absolute determination of MDV1 andHVT genome copy in qPCR assays. Both assays show gooreproducibility and can reliably detect as few as 5 copies of pAI-meq plasmid and 75 copies of pAI-sorf1 plasmid DNA per reac-tion. Because these lower limits of detection were determinedin a 10-fold dilution series they are conservative estimates, withthe true limit of detection lying between this value and the 1:10dilution of them which did not amplify. The lower sensitivityof the HVT assay was reported previously before conversion otM entlp

thep om0 , indc se s ov ds tov ndH ciaten DVD con

version of previous standards has overcome this problem andprovides certain other advantages. It provides standards con-taining a mixture of viral and host DNA as do the unknownsamples. Moreover, our experience is that the reference standardis more stable than the plasmid standard particularly at low con-centrations. It is also preferable to use the infected tissue-derivedstandard to reduce the risk of contamination of the laboratoryenvironment.

We have previously reported on the application of theseassays to infective spleen material (Islam et al., 2005a) and alsoreported more detailed analysis of patterns of viral load in poul-try dust from the field (Walkden-Brown et al., 2005) and fromisolators (Islam et al., 2005c). This paper confirms that the abilityof the assays to quantify MDV1 and HVT in dually infected PBL,feather tip samples and field dust samples. The MDV1 genomecopies measured in feather tips and PBL are in broad agreementwith recent reports (Baigent et al., 2005). MDV1 loads havebeen shown to be higher in feather follicle epithelium than PBLand spleen cells (Davidson and Borenshtain, 2003; Baigent etal., 2005).

To express the viral copy number per million host cells, wedivided the total amount of DNA in the template (25 ng) by thequantity of DNA from a chicken diploid cell (2.5 pg) reported byGregory (2005). This method requires (a) that the DNA amountin the template be accurately measured, (b) that it comprise pre-dominantly chicken genomic DNA and (c) that the extractione ls.O sat-i siblef ainstt1 oul-t unityf ari-a son,w its ofc thodf p-t r allq mate-r n sot roge-n ned.G hostD enc perrg ctionl pieso ec

andH lowt epro-d ngeo ly top ssays

he assays to absolute quantification (Islam et al., 2004). TheDV1-specific qPCR assay was more sensitive than a recublished assay that could detect 44 copies (Baigent et al., 2005).

The plasmid standard curve exhibited parallelism withrevious standards (based on dilution of total DNA fr.0025–250 ng per reaction) from infected spleen samplesating the same behavior over a 104–105 dilution range. Thinabled the quantitation of the previous standards in termiral copy number and conversion of the previous standariral copy number, allowing absolute quantitation of MDV1 aVT genome copy in future and past assays. The cell-assoature of MDV has prevented the preparation of cell-free MNA to use as a standard in real-time PCR. However, the

d

f

y

i-

f

d

-

fficiency of MDV and host DNA is similar from infected celur quantification of extracted DNA is highly reproducible

sfying the first criterion. The second criterion appears plauor chicken tissues given the size of the chicken genome aghat of a virus such as MDV (approximately 2.4× 106 kbp and60–180 kbp, respectively). However, in the case of field p

ry dust this assumption cannot be made, given the opportor microbial and plant DNA to constitute a significant and vble proportion of the DNA in a dust samples. For this reae have chosen to express the viral content of dust in unopy number/mg of dust. There is no readily available meor determining the validity of the criterion of third assumion but it appears to be plausible and indeed it is made foPCR assays of this type. Both the spleen and feather tipial are heterogeneous in terms of their cellular compositiohe copy number obtained is an average per cell in a heteeous population. In the case of PBL, the cell type is defiiven that each reaction in our assay contains 25 ng ofNA, this is the equivalent of the DNA from 10,000 chickells. A limit of MDV1 assay detection of 5 plasmid copieseaction is equivalent to 2.5 copies of MDV genome (as themeqene has 2 copies) per 10,000 cells. Similarly assay dete

imit of 75 plasmid copies per reaction corresponds to 75 cof HVT genome per 10,000 cells as theSorf1 gene is a singlopy.

In summary, we have described and validated MDV1VT qPCR assays providing absolute quantification with

hresholds of detection, a wide dynamic range and good rucibility. They have been shown to have application for raf samples from the host and environment and are likerove essential tools in MD research and control. These a

A. Islam et al. / Journal of Virological Methods 132 (2006) 127–134 133

should play an integral role in the development of more effec-tive disease monitoring and management systems to monitorvaccination performance and reduce the impact of pathogenicMDV1 in the chicken industries.

Acknowledgement

This work was financially supported by the AustralianResearch Council (LP0211607), Baiada Poultry Pty Ltd. andBioproperties Pty. Ltd. for which we are grateful. We also thankDr. A.F.M. Fakhrul Islam, Ms. Sue Burgess, Ms. Megan Suther-land, Mrs. Jenifer Druitt, Dr. Greg Underwood, Dr. Peter Grovesand Dr. Zhanhai Gao for provision of advice, samples and tech-nical support.

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B . In:ol.

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W rzey,aseAust.

W lam,oni-: Nair,earch:sium.

itute

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