journa l homepage: www.e lsev ier .com/ locate / jv i romet
evelopment of a short oligonucleotide microarray for the detection anddentification of multiple potyviruses
ing Weia,∗, Michael N. Pearsona, Dietmar Blohmb, Manfred Nöltec, Karen Armstrongd
School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New ZealandCentre of Applied GenSensor Technology, University of Breme, FB2-UFT, 28359 Bremen, GermanyCentre of Technomathematics, University of Bremen, FB3-TeZeM, 28359 Bremen, GermanyBio-Protection Research Centre, Lincoln University, Canterbury, New Zealand
rticle history:eceived 20 April 2009eceived in revised form 21 July 2009ccepted 27 July 2009vailable online 3 August 2009
eywords:ligonucleotide microarrayrobe
a b s t r a c t
The genus Potyvirus is the largest and one of the most economically important virus genera infectingplants. However, current diagnostic techniques are limited in their ability to identify multiple potyvirusinfections. An assay that can identify multiple potyviruses simultaneously, with good specificity andsensitivity, is therefore highly desirable. To determine the feasibility of simultaneous detection of multi-ple potyviruses a 25-mer oligonucleotide microarray was developed targeting four distinct potyviruses:Dasheen mosaic virus (DsMV), Leek yellow stripe virus (LYSV), Potato virus Y (PVY) and Zucchini yellow mosaicvirus (ZYMV). A total of 85 probes including 33 perfect-match and 52 mismatch probes were designedfrom conserved and variable sequence regions of the nuclear inclusion b (NIb) gene, RNA-dependent RNA
′
otyvirus polymerase (RdRp) gene, coat protein (CP) gene and the 3 untranslated region (UTR), representing the
four targeted potyviruses at both species and strain levels. Each probe was synthesized with spacers ofeither 6 or 12 poly-cytosine or poly-thymine at the 5′ terminus. The array showed high specificity whentested with nineteen different geographically diverse potyvirus isolates of the four target species, fourdistinct but closely related potyviruses, and four healthy plant species. The approaches and protocols
orm ato ge
developed in this study fprovide useful insights in
. Introduction
Potyviruses were established as a taxonomic group in 1959Brandes and Wetter, 1959). Of the six genera in the Potyviridaeamily, Potyvirus is the largest and the type species is Potato virus YPVY). Potyvirus is also the largest and one of the most rapid growingf the 78 plant virus genera (Fauquet et al., 2005). Viruses belong-ng to this genus are transmitted mechanically and by aphids in aon-persistent manner and are responsible for severe disruptionso many economically important crops (Fauquet et al., 2005; Shuklat al., 1994).
Accurate diagnosis is crucial for the effective control of potyvirusiseases. However, the precise detection and identification oftenequires multiple techniques, including symptomatology, host
ange, serology, RT-PCR and sequencing. Reasons for this include:1) the diversity of potyviruses, some with a wide range of hostsnd/or a lack of vector specificity (Fauquet et al., 2005; Shukla et al.,994); (2) serological cross-reactions between related viruses (Lana
et al., 1988; Shukla et al., 1992) or non-reciprocal serological reac-tions between pairs of viruses (Hollings and Brunt, 1981; Shuklaand Ward, 1989); (3) mixed infections of different virus speciesand/or strains in a single plant (Takaichi et al., 2001; Wintermantel,2005; Lorenzen, 2006), plus the high frequency of recombinantsin potyviruses (Chare and Holmes, 2006; Revers et al., 1996); (4)RT-PCR fails to detect all sequence variants and/or species, or ampli-fies preferentially some viruses in mixed infections, thus missingsome components of the mixture. Therefore a simple, rapid, reli-able assay which can detect a range of different potyvirus speciesor strains simultaneously, with good specificity and sensitivity, ishighly desirable.
DNA microarrays have been in use for two decades (Bains andSmith, 1988) and are now considered a convenient and powerfultool for parallel, high-throughput experimentation in molecu-lar biological research (Shoemaker et al., 2001). Oligonucleotidemicroarrays (Saiki et al., 1989) are high density arrays that exhibita number of advantages over cDNA arrays: (1) there is greater con-
trol over the design because oligonucleotide probes can be designedbased on all available sequence information; (2) the arrays can beconstructed with higher density and complexity, especially whenarrays are synthesized in situ by photolithography (light-directedcombinational synthesis or ink jet type methods); (3) the arrays
ave more uniform physicochemical characteristics and there areewer issues pertaining to cross-hybridization; (4) the ease of inilico design and the high specificity of oligonucleotides enablehe discrimination of the mixed infection by different pathogensr even similar strains; (5) in addition, previously recognized dis-dvantages of oligonucleotide arrays, such as lower sensitivity origher background noise, have been improved or overcome by
nnovative solutions in chemistry (Todt and Blohm, 2009), sys-ems engineering and revolutionary techniques (Graves et al., 1998;
atysiak et al., 1999; Nagino et al., 2006).Because of these advantages, oligonucleotide arrays have the
otential to fulfil the requirements of accurate disease diagnosisnd are now becoming powerful diagnostic tools for human (Laassrit al., 2003; Park et al., 2001) and animal diseases (Khadijah etl., 2003; Martín et al., 2006). Boonham et al. (2003) introducedcDNA microarray for the detection of several plant viruses infect-
ng potato crops. Bystricka et al. (2003) and Lee et al. (2003) alsoeveloped cDNA arrays for detection of different viruses infect-
ng potato and cucurbit, respectively. Subsequently Bystricka etl. (2005) reported the first synthetic plant oligonucleotide (40-er) microarray able to detect six potato viruses at both species
nd strain level. These plant disease detection microarrays tar-eted several different viruses within the same host and showeddvantages over the conventional methods such as serology andCR techniques.
The aim of this study was to look at the potential of shortligonucleotide arrays for the detection and identification of multi-le potyviruses at both species and strain levels. In addition factorsffecting array hybridization efficiency were considered duringoth in silico design and testing periods in order to obtain an arrayith high specificity and sensitivity.
. Materials and methods
.1. Potyvirus identification
Thirty different suspected potyvirus infected plant samples, rep-esenting nineteen plant species, were collected in New Zealandnd tested using serology and RT-PCR. Virus identity was provision-lly confirmed using four potyvirus universal degenerate primersn various combinations: (1) primers U335 and D335 (Langeveldt al., 1991) which amplify a ∼0.335 kb fragment from core CPegion of members of the Potyvirus genus; (2) primers PV2I/T7 andV1/SP6 (Mackenzie et al., 1998) which amplify a ∼1.6–2.1 kb frag-ent covering the partial NIb gene, the complete CP gene and 3′
TR of viruses belonging to the Potyviridae; (3) primer pair PV2I/T7Mackenzie et al., 1998) and D335 (Langeveld et al., 1991) thatmplify a ∼1.3 kb fragment of partial NIb and CP genes; and (4)rimer pair U335 (Langeveld et al., 1991) and PV1/SP6 (Mackenziet al., 1998) that amplify a ∼0.7–1.0 kb fragment of partial CPene and 3′ UTR. The last two primer combinations amplify frag-ents with a ∼0.335 kb overlap. PCR reactions were performed in a
5 �l reaction mix containing 10 mM Tris–HCl, 50 mM KCl, 2.5 mMgCl2, 0.2 mM dNTP, 1 unit AmpliTaq DNA polymerase (Applied
iosystems, Scoresby, VIC, Australia), and 0.5 �M of each primer.he reaction parameters for primers U335 and D335 were: 94 ◦Cor 3 min, 30 cycles of 94 ◦C for 30 s, 53 ◦C for 30 s, and 72 ◦C for0 s, followed by a 6 min extension at 72 ◦C and 94 ◦C for 3 min;hose for primers PV2I/T7 and D335, U335 and PV1/SP6, PV2I/T7nd PV1/SP6 were 30 cycles of 94 ◦C for 45 s, 56 ◦C for 45 s, 72 ◦C formin and 30 s, followed by a 6 min extension at 72 ◦C. PCR products
ere separated by electrophoresis on 1% agarose-TBE gels and visu-
lized under UV light after staining with ethidium bromide (finaloncentration 0.5 �g/ml).
Purified amplicons were cloned in the vector pGEM-T EasyPromega Corporation, Madison, WI, USA) according to the man-
ethods 162 (2009) 109–118
ufacturer’s instructions and recombinant plasmids were extractedusing FastPlasmid Mini Kit (Eppendorf, Hamburg, Germany). Thecloned inserts were sequenced with primers to the vector sequenceusing a 3130XL capillary sequencer (Applied Biosystems, Mel-bourne, Australia) and the sequences compared with those inGenBank using the Basic Local Alignment Search Tool (BLAST) pro-gram (Altschul et al., 1997).
2.2. Selection of suitable genome regions for probe design
Based on the published literature the following five regionsof the Potyvirus genome were identified as potentially containingboth unique conserved sequences to identify individual potyvirusspecies and variable sequences to identify different strains withineach species: 5′ UTR (Kekarainen et al., 1999; Simón-Buela et al.,1997), P3 gene (Aleman-Verdaguer et al., 1997), NIb gene (Gibbsand Mackenzie, 1997), CP gene (Bateson et al., 2002; Jordan, 1989;Tsuneyoshi et al., 1998) and the 3′ UTR (Frenkel et al., 1989; Uyeda,1992). Sequences for these five regions of the targeted Potyvirusspecies were downloaded from GenBank and aligned using BioEditversion 7.0.4 (Hall, 1999) for the identification of conserved andvariable sequence regions.
2.3. Probe design, selection and modification
Both conserved and variable sequences were used for probedesign using two different software packages: ROSO (Reymondet al., 2004) and a software package developed by the bioinfor-matics group of the CAG, University of Bremen, Germany (Nölte,2002; Kochzius et al., 2008). To increase the likelihood of identifyingvirus specific probes: (1) multiple probes were designed from theselected genome regions of each target virus; (2) during the designprocess probes were delimited against 549 sequences representing80 Potyvirus species and four plant genomes (Oryza sativa chro-mosome [accession no. AC148235], Beta vulgaris subsp. vulgarismitochondrion [accession no. NC 002511], Saccharum hybrid cul-tivar sp-80-3280 chloroplast [accession no. NC 005878], Nicotianatabacum plastid [accession no. NC 001879]); (3) selected probeswere BLASTed against all nucleotide sequences in GenBank in June2005 to identify the highest identity to species specific (SpS) orNew Zealand strain specific (StS) regions. Human gene sequences(accession no.: NM 000979 and NT 011109) were used for design-ing negative control probes using the same design and selectionprocedures. Two positive control sequences to indicate hybridiza-tion efficiency (with no relationship to Potyvirus sequences) wereprovided by CAG, University of Bremen, Germany.
To evaluate their effect on hybridization efficiency 6 or 12 cyto-sine or thymine were added to the 5′ end of each probe. Theyincrease additionally the space between probes and the slide sur-face irrespective of the 6-carbon units supporting the terminalamino group which attaches the oligonucleotides to the slide sur-face.
2.4. Array production and validation
2.4.1. Array production and hybridizationThe oligonucleotide probes were synthesized by ILLUMINA Inc.,
San Diego, CA, USA, and printed onto glass slides by CAG (Universityof Bremen, Germany) at a density of 4.5 × 1011 molecules/cm2.
Three different sizes of PCR products ∼0.335 kb, ∼0.7–1.0 kband ∼1.3 kb, which were amplified from the targeted potyvirus
species using the primer pairs and PCR conditions described in Sec-tion 2.1, were used to hybridize with arrays. PCR products werelabelled with fluorescent Cyanine dyes Cy5 or Cy3 (Amersham Bio-sciences, Rydalmere, NSW, Australia) using both direct and indirectmethods. Direct labelling used a Cy5 labelled reverse PCR primer
T. Wei et al. / Journal of Virological M
Table 1Overseas potyvirus isolates used to validate the microarray.
Potyviruses Source of isolate Company
Dasheen mosaic virus Not specified AgdiaUSA (FL) DSMZ
Thermo Electron GmbH, Karlsruhe, Germany) to label the anti-ense strands of the target DNA. Indirect labelling, using a dNTPixture of aminoallyl- and aminohexyl-modified nucleotides from
uperScriptTM Indirect cDNA Labelling System (InvitrogenTM Lifeechnologies, Auckland, New Zealand), enabled the PCR productso couple with a fluorescent Cy5 or Cy3 dye by the SuperScriptTM
ndirect cDNA Labelling System (InvitrogenTM Life Technologies,uckland, New Zealand). Two Cy5 labelled positive targets withomplementary sequences to the two positive control probesrovided by CAG, and which had no relationship to Potyvirusequences, were used to show the efficiency of the hybridization.
The hybridization mixture was prepared in a total volume of0 �L containing 10 nM of target viral PCR product, 1 nM positiveontrol targets, 1× liquid blocking reagent (Amersham Biosciences,ydalmere, NSW, Australia), and 1× hybridization buffer (20 mMris–HCl, pH 7.3; 150 mM NaCl; 5 mM EDTA, pH 7.3; 0.05% Tween-0; 0.045% milk-powder; 0.01 mg/mL salmon sperm DNA). Theybridization mixture was denatured at 95 ◦C for 5 min, placed
mmediately on ice for 3 min and then applied to the array slides.fter incubation at 55 ◦C for 2 h, the slides were washed twice inETBS buffer (TBS buffer plus 5 mM EDTA and 0.05% Tween-20)nd then twice in TBS buffer (20 mM Tris–HCl, pH 7.3 and 150 mMaCl).
Additionally, PCR products used to assess the specificity of therray were generated from a total of fifteen overseas isolates ofhe four targeted Potyvirus species from nine different countriesTable 1) plus four distinct but closely related potyvirus selected
rom species found in New Zealand (Table 2). Indirectly labelledDNAs synthesized from RNA isolated from a range of healthylants using random hexamer primers (Invitrogen, Auckland, Newealand) were also used to validate the arrays.
able 2ew Zealand potyviruses identified in this study.
The concentration of target fragments was calculated using fol-lowing formula:
1 pmol dsDNA ≈ the length of DNA fragment × 2 × 330×10−3 ng (1 base ≈ 330 g/mol)
1 pmol ssDNA ≈ the length of DNA fragment × 330 × 10−3 ng (1 base ≈ 330 g/mol)
2.4.2. Sensitivity of the arrayTo evaluate the sensitivity of the array, selected target DNAs at
a range of dilutions were hybridized with the array.
2.4.3. Data analysisThe array images were analyzed using GenePix® Pro 5.0 (Axon
Instruments Inc., Concord, ON, Canada) and statistical analysis wasconducted using Microsoft EXEL and Minitab Software (release 15,PA, USA).
The criteria for determining positive and negative thresholds forthe microarray spots can potentially be based on both visual assess-ment and fluorescent intensity values. For the latter the thresholdfor positive results was set at the mean plus 4× standard devi-ation (SD) of the fluorescent intensity values of negative controlspots. This was a conservative threshold chosen to avoid false pos-itives from weak non-specific cross-reaction and correlated wellwith visual assessment of the fluorescent image.
3. Results
3.1. Potyvirus identification and selection of suitable potyvirusfor array development
During the survey of New Zealand potyviruses thirteen differentPotyvirus species were identified from suspected infecting plants(Table 2). Of these, four phylogenetically distinct potyviruses wereselected for a proof of concept study based on the availability ofmultiple sequences in GenBank to represent the diversity of eachspecies. These were: Dasheen mosaic virus (DsMV) from Coloca-sia esculenta (taro), Leek yellow streak virus (LYSV) (two distinctisolates) from Allium sativum (garlic), Potato virus Y (PVY) fromSolanum tuberosum sp. (potato), and Zucchini yellow mosaic virus(ZYMV) from Cucurbita pepo subsp. pepo (Zucchini).
3.2. Selection of suitable genome regions for probe design
Analysis of a total of 586 sequences from GenBank, represent-ing five selected genome regions of DsMV (42 sequences), LYSV(58 sequences), PVY (265 sequences) and ZYMV (221 sequences),identified only a few sequences with short conserved regions forthe 5′ UTR region and P3 gene. Therefore these two regions werenot considered suitable for probe design. In contrast the NIb gene,CP gene and 3′ UTR region of the four selected potyvirus specieswere found to contain several conserved (species specific) and dis-tinct (strain specific) sequences of the desirable length. In additionthese regions can be amplified using existing universal potyvirusprimers. Therefore these three regions were considered suitable fordesigning the probes.
3.3. Probe design, selection and array production
Sense strand sequences of New Zealand DsMV (AY994104),LYSV (AY842134 and AY842136), PVY (DQ217931) and ZYMV(AY995214) containing the three target regions were used to design
the probes to detect either PCR products (anti-sense strands) orvirus cDNAs. A preliminary analysis of different lengths of probesshowed that a length of 25-mer was optimal to generate a suffi-cient number of potential probes from all three genome regions(Table 3). A total of 85 potyvirus probes (Table 4), including 33
112 T. Wei et al. / Journal of Virological Methods 162 (2009) 109–118
Table 3The number of potential probes of different lengths generated by ROSO.
Virus/genome region 70-mer 60-mer 50-mer 45-mer 40-mer 35-mer 30-mer 25-mer
erfect-match (PM) and 52 mismatch (MM) probes were selected
s follows: DsMV (9 PM and 13 MM), LYSV (10 PM and 18 MM),VY (6 PM and 8 MM) and ZYMV (8 PM and 12 MM). The MMrobes contained 1–9 nucleotide mismatches to the experimen-al New Zealand virus isolates but were a perfect-match to one or
able 4otal 85 probes developed for microarray synthesis (name in bold representing the probesismatch probe; probe sequence bases in bold representing mismatch-oligonucleotides)
more other isolates of the same species from GenBank. Of these
probe sequences, 26 and 59 probes were generated by CAG andROSO, respectively. In addition, five negative control probes of thesame length were designed based on human sequences. As eachprobe was also produced with four different spacer modifications
synthesized by CAG software from Bremen University; name with “m” representing.
T. Wei et al. / Journal of Virological Methods 162 (2009) 109–118 113
Fig. 1. Probe printing pattern. White spots = Potyvirus probes; black spots = negativecontrol (NC) probes; red spots = positive control (PC) probes; blue spots = emptyspots. The spots representing different potyviruses and NC probes are separatedbs2
(s
3
3a
uZotpoCwbatsmPwsifls
Table 5Hybridization results using PCR products from NZ potyviruses.
Fig. 3. Fluorescent intensities of probes without spacer and with 12T spacer from a
Fu
y yellow lines. The five set of probes with and without spacer modifications areeparated by black lines. Each set of probes contains 85 Potyvirus probes, 5 NC probes,PC probes and 6 empty spots.
6C, 12C, 6T or 12T) a total of five sets of 90 probes were finallyynthesized. The array printing pattern is shown in Fig. 1.
.4. Specificity of the array
.4.1. Hybridization of New Zealand isolates of DsMV, LYSV, PVYnd ZYMV
Initial hybridizations using the three different sizes of PCR prod-cts (∼0.335 kb, ∼0.7–1.0 kb and ∼1.3 kb) from sequenced Newealand isolates of DsMV, LYSV, PVY and ZYMV resulted in onlyne PCR product from each of the four-virus species giving a posi-ive hybridization result (Table 5). In total 26 probes (30.6%) gave aositive signal including 14 PM (out of 33) and 12 MM probes (outf 52). Of these positive probes, 13 (out of 26) were designed byAG and 13 (out of 59) by ROSO. No cross-species hybridizationas observed. Similar hybridization results were obtained with
oth labelling systems except that (a) indirect labelling providedhigher fluorescence signal for most positive probes (except for
hose with pixel saturation) and (b) one additional weak positiveignal was obtained from the indirect-labelled ∼1.3 kb DsMV frag-ent (Figs. 2 and 3). When the four PCR products from DsMV, LYSV,
VY and ZYMV (∼1.3 kb, ∼1.0 kb, ∼0.8 kb and ∼0.7 kb, respectively)ere hybridized in a mixture, the positive patterns were exactly
ame as those from individual hybridizations (Figs. 4 and 5). Pos-tive probes with spacers, especially 12C or 12T, showed higheruorescent intensities than those without spacers or with shorterpacers (Figs. 2–5).
hybridization using direct- and indirect-labeled DsMV ∼1.3 kb fragment. The heightof the peaks represents the fluorescent pixel intensity of positive spots with thebackground subtracted. The numbers adjacent to the peaks are the probe identifi-cation numbers. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of the article)
ig. 2. Microarray results from hybridization of the DsMV ∼1.3 kb fragment. Image (A) hybridization using direct-labelled fragment with Cy5 dye. Image (B) hybridizationsing indirect-labelled fragment with Cy5 dye. The numbers to the left or lower-left side of the positive spots are the DsMV probe identification numbers.
114 T. Wei et al. / Journal of Virological M
Fig. 4. Microarray results from hybridization with a mixture containing PCR prod-ucts from four potyviruses: direct-labelled DsMV ∼1.3 kb fragment with Cy5 dye,indirect-labelled LYSV ∼1.0 kb fragment with Cy3 dye, direct-labelled PVY ∼0.8 kbfragment with Cy5 dye and indirect-labelled ZYMV ∼0.7 kb fragment with Cy3. Thenumbers to the left or lower-left side of the positive spots are the probe identificationnumbers.
Fig. 5. Fluorescent intensities of probes without spacer and with 12T spacer froma hybridization using four-virus mixture. The height of the peaks represents thefluorescent pixel intensity of positive spots with background subtracted. The num-bers adjacent to the peaks are the probe identification numbers from relevantpotyviruses. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of the article)
Table 6Probes reacting positively with overseas potyvirus isolates compared with New Zealand i
a “m + a number” following the probe number indicates the number of mismatched nu
ethods 162 (2009) 109–118
3.4.2. Hybridization of worldwide isolates of DsMV, LYSV, PVYand ZYMV
Given the similar results obtained with both direct and indirectlabelling methods, direct labelling was selected for further testingas it is more convenient and less expensive. Directly Cy5 labelled∼0.7 kb and ∼1.3 kb PCR products were generated successfully fromfourteen isolates of the four targeted Potyvirus species and werehybridized with the arrays (PCR failed for one Italian ZYMV iso-late). All PCR products, except those from the South America PVYisolate, gave similar hybridization results to the New Zealand iso-lates (∼1.3 kb product from DsMV, ∼1.0 kb product from LYSV, and∼0.7 kb products from PVY and ZYMV showed positive spots). Theoverseas PVY isolates reacted with exactly the same probes as theNew Zealand isolate although the signals of some probes wereweaker. The other viruses shared some positive probes in commonwith New Zealand isolates (although the fluorescent intensity ofsome probes varied between the different isolates) but also gavesome differential reactions with other probes (Table 6). Resultsbased on the analysis of fluorescent intensity values were the sameas those from visual assessment except for one Netherlands PVYprobe that was not visually detected but showed a positive flu-orescent intensity value (data not shown). Only one non-specifichybridization event was observed, the ∼1.0 kb fragment of theNetherlands LYSV reacting positively to a ZYMV probe (data notshown).
Cloning and sequencing of the non-reacting South America PVYisolate (GenBank accession no. EF488081) showed that it shared94.2% nt identity to Pepper yellow mosaic potyvirus (AF348610) andonly 43–51.5% nt identity to PVY isolates, thus explaining the neg-ative result.
3.4.3. Hybridization of closely related potyviruses and healthyplant cDNA
Four non-target but closely related potyviruses were identi-fied by creating a neighbour joining tree using 21 New Zealandpotyvirus species and analysis by Mega 3.1 (Kumar et al., 2004).These were: Ornithogalum mosaic virus (OrMV) and Ornithogalum
virus 2 (OrV2) which were closely related to LYSV, Onion yellowdwarf virus (OYDV) that is closely related to PVY, and Zantedeschiamild mosaic virus (ZaMMV) that is closely related to both DsMVand ZYMV. When indirectly labelled ∼1.3 kb and ∼0.7 kb PCR prod-ucts from these four viruses were hybridized with the microarray,
cleotides in that probe. Numbers in bold are probes designed by the CAG software.
T. Wei et al. / Journal of Virological Methods 162 (2009) 109–118 115
F ix differ
nen
hCfn
3
∼twm0tcpatnoepopw
4
rti
ig. 6. Microarray results from the hybridization of the ZYMV ∼0.7 kb fragment at sepresent ZYMV probe numbers.
one of the fragments gave a positive result, as determined byither visual assessment or the fluorescent intensity value (dataot shown).
Indirectly labelled cDNA from total RNA preparations of fourealthy plant species: N. benthamiana, Cucurbita pepo (zucchini),olocasia esculenta (taro) and the grass Dactylis glomerata (cocks-oot) were also hybridized with the microarray and all gaveegative results (data not shown).
.5. Sensitivity of the array
Based on the initial array results, the direct-labelled ZYMV0.7 kb fragment was selected to test the potential sensitivity of
he arrays as 3–5 times higher concentrations of PCR productsere obtained in a 25 �L reaction than other three positive frag-ents. Six different final concentrations (30 nM, 10 nM, 2 nM, 1 nM,
.5 nM and 0.1 nM) of the target fragment were hybridized withhe microarrays. The results showed that increasing the targetoncentration to 30 nM did not result in any additional positiverobes when compared to the original hybridization concentrationt 10 nM, but higher concentrations did give higher signal intensi-ies for all the positive spots. Below 10 nM both the intensity andumber of positive probes declined, especially for the probes with-ut a spacer, but even at the lowest concentration of 0.1 nM (whichquates to an original cDNA concentration of ∼3.2 atograms), threeositive spots were still visually discernable (Figs. 6 and 7). Basedn the fluorescent intensity value range of the strong and weakositive probes from other viruses at 10 nM, a similar sensitivityould be expected.
. Discussion
Microarrays have become increasingly important in both basicesearch and diagnostics. Plant potyviruses are particularly difficulto identify precisely by serological tests such as ELISA since serolog-cal cross-reactions are quite common (Du et al., 2006; Jaegle and
rent concentrations. The numbers to the left or lower-left side of the positive spots
Van Regenmortel, 1985; Shukla et al., 1992). Similarly, PCR with-out sequencing can lead to misidentifications (Okuda and Hanada,2001; Raut and Kapadnis, 2007). The purpose of this study was todetermine whether it was feasible to develop a microarray for plantpotyviruses that could specifically detect target potyvirus speciesand potentially distinguish different strains within the species.
A microarray was developed for four potyvirus species whichshowed high species specificity when tested using a range ofisolates from the target species, plus four other closely relatedpotyviruses, and four healthy plant species.
There are many factors that can affect the specificity and sen-sitivity of a microarray. An ideal oligonucleotide probe shoulddiscriminate clearly between its intended target fragment and allother non-related targets in a selected pool (Nielsen et al., 2003).A critical factor is the possibility of cross-hybridization betweenoligonucleotide probes and non-target templates but completediscrimination is often difficult to achieve (Zhang, 2004). Kaneet al. (2000) reported cross-hybridization when the “non-target”sequences had more than 75–80% identity with the probes and/orwith a homologous region was more than 30% of the length of theprobe. Thus, not only the total homology percentage of sequences,but also the length of homologous regions should be considered.In order to overcome or minimize non-specific hybridizations andobtain as many reliable probes as possible, two strategies wereapplied in this study. The first strategy was to set the selectioncriteria in the two different probe design programs (CAG andROSO) to eliminate those target sequences with >75–80% identity tosequences in the delimitation pool of 549 sequences representing80 potyviruses and four complete plant genomes. The second strat-egy was a second round of selection by individually comparing eachprobe sequence generated by the software against GenBank using
BLAST. Consequently the final selected probes had a high specificityto the targeted species or strains. Based on these strategies, a totalof 85 probes including 26 CAG probes and 59 ROSO probes wereincluded in the microarray. The hybridization results, using NewZealand isolates, showed that the CAG software followed by BLAST
116 T. Wei et al. / Journal of Virological M
Fig. 7. Fluorescent intensities of probes without spacer and with 12T spacer froma hybridization using the direct-labelled ZYMV ∼0.7 kb fragment at six differentconcentrations. The height of the peaks represent the fluorescent pixel intensity ofptc
st
ep
microarray hybridization (Evertsz et al., 2001; Kane et al., 2000).
ositive spots with background subtracted. The numbers adjacent to the peaks arehe probe identification numbers of ZYMV. (For interpretation of the references toolor in this figure legend, the reader is referred to the web version of the article)
election generated a higher percentage of positive probes (50.0%)
han the ROSO software (22.1%).
Due to the extremely high sequence variability between gen-ra and species of Potyviridae, the design of oligonucleotide probesresents difficulties at the genus, species and strain levels. At
ethods 162 (2009) 109–118
the genus level, only a few conserved regions could be found.This is confirmed by the fact that the commonly used uni-versal potyvirus primers (Langeveld et al., 1991; Mackenzie etal., 1998) contain up to 38% degenerate bases. At the specieslevel, probe design is exacerbated by the limited sequencesavailable for many species. Consequently, in this study, an impor-tant criterion for the experimental potyviruses was a sufficientnumber of sequences available in GenBank to represent the diver-sity of the virus. This allowed the design of multiple probesto several regions of each target virus and thus enhanced theprobability of successful detection. To minimize the impact ofsequence variability and allow the design of multiple probes foreach virus, a probe length of 25-mer was shown to be opti-mal.
In many studies searching for parameters affecting the microar-ray hybridization efficiency, steric hindrance mediated by the solidsupport was considered an important factor (Shchepinov et al.,1997). Spacer molecules, either on the probe or on the surface ofthe solid support, play an important role in improving the bindingproperties and hybridization efficiencies (Graves et al., 1998) byextending the probe away from the solid surface, thus reducing thesteric hindrance. Shchepinov et al. (1997) reported that the use ofspacer molecules of a minimum length of 40 atoms could increasehybridization yield up to 150-fold. Single units of hexaethylene gly-col (Maskos and Southern, 1992) or hexa-ethyloxy-glycol (Chou etal., 2004) and a variety of monomeric units using phosphoramiditechemistry (Shchepinov et al., 1997) have been commonly used asspacers. In addition, with poly-adenine (polyA) spacers, increasingthe spacer length from 6-mer to 24-mer is reported to enhance sig-nal intensity (Peplies et al., 2003). In this study a standard aminomodifier containing a 6-carbon linker arm, was positioned at the5′-end of all the probes with either 0, 6 or 12 cytosine or thyminespacers. The results showed that all probes with spacers producedstronger signals than those without a spacer and in general probeswith a twelve residue spacer (either C or T) gave stronger signalsthan probes with six residue spacers, although in most of the casesthe differences were not statistically significant (p > 0.05). This maybe due to the variable fluorescent intensity values obtained fromthe triplicate experiments which often resulted in a high standarderror.
The labelling system is another major factor affecting microar-ray hybridization efficiency (Grigorenko, 2002). Xiang et al. (2002)and Bystricka et al. (2005) considered that indirect labelling ofcDNA was more effective and less expensive than direct labelling.However, in this study, in which used PCR products were used,both labelling systems provided almost identical positive/negativehybridization patterns, although indirect labelling provided gen-erally higher fluorescent intensities and slightly higher sensitivitythan direct labelling. However, direct labelling was consideredcheaper, easier and quicker than indirect labelling.
In addition to specificity, sensitivity is a crucial factor in diagnos-tic tests. In this study, using a combination of PCR and microarrays,it was possible to detect ∼3.4 ng of PCR product which was equiv-alent to ∼3.2 atograms of the original viral cDNA. This detectionsensitivity is much higher than that reported for other microarrayswhich required 1–100 �g of total RNA (Kern et al., 2004; Roth etal., 2004) or 5 pg of genomic DNA (Szemes et al., 2005) or 500 fgof pathogen DNA (Wilson et al., 2002). The higher sensitivity of thearray developed in this study may be due to the addition of cytosineor thymine spacer to the probes.
Non-specific cross-hybridization is a well known problem for
In this study only one case of non-specific cross-hybridizationwas observed: a single LYSV isolate reacted to a ZYMV probe.Although it is not difficult to identify the true positive results byanalysing of the multiple probes for each virus, this highlights
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he importance of using multiple probes for each virus so thathe occasional non-specific reaction can be seen to be an aberrantesult, and if necessary those probes can be eliminated from therray.
The PCR amplification step failed for one potyvirus (ItalianYMV). This may have been due to sequence variability at therimer binding sites, wrong identification of the virus or possiblyue to degradation of the sample during transit or storage.
Although the use of microarrays has increased rapidly over theast 15 years, most of the factors affecting the DNA duplex for-ation are still far from clear (Peplies et al., 2003; Southern et al.,
999). It has been observed commonly that some perfect-matchargets and probes fail to hybridize (Loy et al., 2002; Southern and
askos, 1994) while some mismatch probes yield higher signalntensities than those of corresponding perfect-match probes (Naefnd Magnasco, 2003). Both of these phenomena were observed inhis study and require further investigation.
In summary, a microarray with high specificity and sensitiv-ty was developed for the detection and differentiation of fourotyvirus species and different strains within each of the species.or viruses with highly variable sequences, such as potyviruses,he approaches used in this study, including short (25-mer) probes,wo rounds of selection, and the addition of spacers, have provedffective in selecting multiple probes from different regions of virusenome with high specificity. Microarrays have previously beenesigned for a limited number of viruses in a single crop, such ashe 40-mer oligonucleotide microarray of Bystricka et al. (2005) foretecting potato potyviruses. However, designing microarrays toetect multiple viruses from multiple crops, such as the wide rangef potyviruses found in the many species of ornamentals, providesfar greater challenge. The approach described in this paper has
he potential for application to microarrays for large numbers ofotyviruses and possibly other viral pathogens of plants and otherrganisms.
cknowledgements
We thank Kelvin Lau, Associate professor Cristin Print, Dr. Franzichler, Dr. Lei Zhang, Dr. Sasha Todt, Mr. Frank Meyerjürgens andr. Sven Roll for their advice and kind help while this projectas in progress and Dr. Robin MacDiarmid for reviewing the draftanuscript. We also thank National Centre for Advanced Bio-
rotection Technologies for providing the funding and the Schoolf Biological Sciences, Auckland University, for providing researchacilities.
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