Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

(1)

ContentslistsavailableatScienceDirect

Journal

of

Virological

Methods

jou rn al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / j v i r o m e t

Analysis

of

sensitivity

and

rapid

hybridization

of

a

multiplexed

Microbial

Detection

Microarray

James

B. Thissen

a

_,

_Kevin

_McLoughlin

b

_,

_Shea

_Gardner

b

_,

_Pauline

_Gu

a

_,

_Shalini

_Mabery

a

_,

Tom

Slezak

b

_,

_Crystal

_Jaing

a,∗

a_Physical_&_Life_Sciences_Directorate,_Lawrence_Livermore_National_Laboratory,_Livermore,_CA_94551,_United_States

b_Computations_Directorate,_Lawrence_Livermore_National_Laboratory,_Livermore,_CA_94551,_United_States

Articlehistory:

Received16August2013

Receivedinrevisedform14January2014

Accepted21January2014

Availableonline3March2014

Keywords: Microarray Pathogen Detection Virus Clinical Environmental

a

b

s

t

r

a

c

t

Microarrayshaveproventobeusefulinrapiddetectionofmanyvirusesandbacteria.Pathogendetection microarrayshavebeenusedtodiagnoseviralandbacterialinfectionsinclinicalsamplesandto evalu-atethesafetyofbiologicaldrugmaterials.AmultiplexedversionoftheLawrenceLivermoreMicrobial DetectionArray(LLMDA)wasdevelopedandevaluatedwithminimumdetectableconcentrationsforpure unamplifiedDNAviruses,alongwithmixturesofviralandbacterialDNAsubjectedtodifferentwhole genomeamplificationprotocols.Inadditiontheperformanceofthearraywastestedwhenhybridization timewasreducedfrom17hto1h.TheLLMDAwasabletodetectunamplifiedvacciniavirusDNAata concentrationof14fM,or100,000genomecopiesin12␮Lofsample.Withamplification,positive iden-tificationwasmadewithonly100genomecopiesofinputmaterial.Whentestedagainsthumanstool samplesfrompatientswithacutegastroenteritis,themicroarraydetectedcommongastroenteritisviral andbacterialinfectionssuchasrotavirusandE.coli.

Accuratedetectionwasfoundbutwitha4-folddropinsensitivityfora1hcomparedtoa17h hybridiza-tion.Thearraydetected2ng(equivalentconcentrationof15.6fM)oflabeledDNAfromaviruswith1h hybridizationwithoutanyamplification,andwasabletoidentifythecomponentsofamixtureofviruses andbacteriaatspeciesandinsomecasesstrainlevelresolution.Sensitivityimprovedbythreeorders ofmagnitudewithrandomwholegenomeamplificationpriortohybridization;forinstance,thearray detectedaDNAviruswithonly20fgor100genomecopiesasinput.Thismultiplexedmicroarrayis anefficienttooltoanalyzeclinicalandenvironmentalsamplesforthepresenceofmultipleviraland bacterialpathogensrapidly.

1. Introduction

Rapid detection and characterization of bacterial and viral

pathogens is important for clinical microbiological diagnostics,

publichealth,drugandfoodsafety,environmentalmonitoringand

biodefense.Variousdetectiontechnologiesbasedonnucleicacid

signatureshaveemergedinthepastfewyears,includingTaqMan

PCRandLuminexbeadbasedsystems.Whilethesetechnologies

are abletoidentify selectedpathogens at thespecies or strain

level rapidly,theydo not have thecapability toprovide broad

∗ Correspondingauthorat:LawrenceLivermoreNationalLaboratory,P.O.Box808,

MailstopL-452,Livermore,CA94551,UnitedStates.Tel.:+19254246574.

E-mailaddress:jaing2@llnl.gov(C.Jaing).

spectrumdetectionaboutknownornovelorganisms.

Character-izationofknown,emerging,engineered,orunknownpathogens

requiresaplatformthathasthecapacitytoassesswholegenome

sequencecontentfromavarietyofpathogensveryrapidly.While

sequencing providesthe most in depth information to

charac-terizeamicrobial genome,thecosts,labor,and timeassociated

withlibrarypreparation,sequencing,bioinformaticanalysis,and

data storage may be prohibitive when analyzing many

iso-lates in a standard laboratory setting. To meet this need, a

pan-microbial microarray was developed, the Lawrence

Liver-moreMicrobialDetectionArray(LLMDA)(Gardneretal.,2010).

The originalversion ofthis array contains 388,000probes

cov-ering all currently sequenced viruses and bacteria, and can

detect any of these organisms within 24h. This microarray

uses long (50–65bp) oligonucleotide probes to enable

detec-tion of novel, divergent species with homology to sequenced

organisms.TheLLMDAhasbeenrecentlyusedtoidentifya

con-taminating pig virus from a rotavirus vaccine (Victoria et al.,

2010),avaccineusedworldwideininfantstopreventrotavirus

http://dx.doi.org/10.1016/j.jviromet.2014.01.024

Open access under CC BY-NC-ND license.

(2)

Table1

Probesummaryfor72KLLMDAarray.

Numberof probes Average numberof probesper sequence Targetsequences

48,893 20 Allviralfamiliesexcept Orthomyxoviridaeandfamily unclassiﬁedcompleteviralgenomes andsegments

7777 10 SegmentsintheOrthomyxoviridae family

2972 10 Familyunclassiﬁedviralgenomesand completesegments

7864 15 Bacterialgenomesandplasmids 3410 – RandomcontrolswithGC%andlength

distributionmatchedtotargetprobes

70,916 Total

infection. This array has also been used to detect viral infec-tions from various human clinical samples (Erlandsson et al., 2011).

Recent development of multiplexed microarrays makes

screeningoflargenumbersofsamplesmorecost-effectivewith

higher throughput(Palka-Santiniet al., 2009; Suo et al., 2010;

Bierbaumet al., 2012).These multiplexed microarrays are low

tomediumdensityandarefocusedonarelativelysmallnumber

ofpathogens.A modiﬁedversion oftheLLMDAwasdeveloped,

using the 4×72K Roche NimbleGen microarray format that

couldserveasabroadspectrumandfastdetectiontechnologyto

analyzebacterialandviralpathogens.Thisarraycontainsatotalof

70,916probes,designedtodetect2200viralspecies(38,000target

sequences) and 900 bacterial species (3500 target sequences).

Atargetwasdeﬁned asaseparately accessioneddraftgenome,

ﬁnishedchromosome,plasmidorviralsegmentsequenced.

Aseriesofexperimentswiththe4×72Karraywasperformedto

estimatetheminimumdetectableconcentrationsofpureviral

tem-platesandmixturesofviralandbacterialtemplates,bothwithand

withoutampliﬁcation,usingvariousrandomampliﬁcation

tech-niques.Thesensitivityandspeciﬁcityofthemicroarraywasalso

exploredwhenitwashybridizedformuchshortertimesthanthe

standard17h.Inadditionthearraywastestedusingknownand

unknownclinicalsamplestoevaluateitsutilityindetecting

infec-tiousagentsfromhumansamples.

2. Materialsandmethods

2.1. Probedesignforthe4×72KLLMDAarray

Probedesignforthe388KversionoftheLawrenceLivermore

MicrobialDetectionArray(LLMDA)wasdescribedinGardneretal.

(Gardner et al., 2010). For the4_×72K version, theinitial pool

ofcandidateprobeswasdown-selectedtohaveatotalof70,916

probes,asindicated inTable1,ratherthan the388,000probes

selectedforthepreviousversion.Eachviralgenomeorsegment

targetwasrepresentedby10–20probesonthe72Karray,in

con-trasttothe50 probespertargetinthe388Kversion.Thesame

processwasusedtodown-selectfromthecandidatepoolof

oli-gosaswasdescribedin(Gardneretal.,2010).Thesamebacterial

probeswereusedasonthe388Kversion,with15probesper

tar-get.Thearrayswerecustomorderedusingthe4-plex72Kformat

fromRocheNimbleGen.

2.2. Nucleicacidextractionfrombacterialandviralsamples

Humanadenovirus type 7 strainGomen (Adenoviridae) and

respiratorysyncytialvirus(RSV)werepurchasedfromtheNational

VeterinaryServicesLaboratory(Ames,IA)andgrownatLawrence

LivermoreNationalLaboratory(LLNL).Forpuriﬁcationof

adenovi-rusDNA,asolutionof10mLvirusculture,1mL5.5%TritonX-100,

and460␮L0.5MEDTAwasaddedtoa50mLtubeandvortexed

vig-orously.Followingvortexing,1.28mL10%SDSand540␮L10U/mL

proteinaseKwasaddedtothemixtureandincubatedat55◦Cfor1h

withmixingevery10min.Afterincubation,420␮L5MNaCland

13.7mL25:24:1phenol/chloroform/isoamylalcoholwasaddedto

thetubeandinverteduntilthemixturewascompletely

homoge-nous.Oncehomogenous,thesolutionwascentrifugedat10,000×g

for10minandtheupperaqueouslayerwastransferredtoanew

tube.Atotalof27.4mLof100%ethanolwasaddedtothe

aque-ouslayerand incubatedat−20◦_C _for₁_h. _Following_incubation,

thesolutionwascentrifugedatmaximumspeedfor10minand

thesupernatantwasdiscarded.Thepelletwaswashedoncewitha

solutionof70%ethanoland150mMNaCl.Theliquidwasremoved

andpelletwasallowedtoair drybeforebeingre-suspendedin

nucleasefreewater.

ForextractionofRNAfromRSVvirus,asolutionof2mLofvirus

cultureand 6mLTRIzol LSReagentfromLifeTechnologies was

mixedvigorouslyandincubated15minatroomtemperature.

Fol-lowingincubation,1.6mLchloroformwasaddedtothecapofthe

tube,shakenvigorouslyfor15s,andincubatedatroom

tempera-turefor15min.Thesolutionwascentrifugedat4◦Cfor15minat

3000×gandtheaqueouslayerwastransferredtoanewtube.70%

isopropylalcoholwasaddedtotheaqueouslayer,mixedby

invert-ingseveraltimes,andincubatedfor10minatroomtemperature.

Thesolutionwascentrifugedatmaximumspeedfor10minat4◦C

andtheliquidwascarefullypouredoff.Theremainingpelletwas

washedwith70%icecoldethanol.Theethanolwaspouredoffand

thepelletwasairdriedbeforebeingre-suspendedinRNasefree

water.

EbolaZaire95andRiftValleyfever(RVF)virusstrainZH501

RNAwereobtainedfromNationalBioforensicAnalysisand

Coun-termeasuresCenter(NBACC).Copynumberquantitatedvaccinia

ListerDNAwaspurchasedthroughAdvancedBiotechnologiesInc.

(Columbia,MD).BacillusanthracisAmesDNAwasobtainedfrom

internalsourcesatLawrenceLivermoreNationalLaboratory(LLNL)

andnucleicacidswereextractedusingtheEpicentreDNA

extrac-tionkitaccordingtomanufacturer’sprotocols.

Extractednucleicacidsamplesfromfourpediatricstoolsamples

fromchildrenwithacutegastroenteritisandenrolledintheNew

VaccineSurveillanceNetwork(NVSN)wereprovidedbyDr.Mike

BowenfromtheCentersforDiseaseControlandPrevention(CDC).

2.3. Nucleicacidampliﬁcation

Thenucleicacidsampleswereampliﬁedusingfourdifferent

protocols: a protocol developed by Wanget al., 2003, referred

tointhisstudyasRandomPCR1;thesameprotocolwith

mod-iﬁed primers (Wong et al., 2007), referred to as Random PCR

2;the TransPlex® _Whole _{Transcriptome}_{Ampliﬁcation} _Kit _(Cat.

WTA1)fromSigmaAldrich(St.Louis,MO) (Iscoveetal.,2002);

andQuantiTectWholeTranscriptomeKit(Cat.207043)from

Qia-gen(Valencia,CA)(Berthetetal.,2008;Erlandssonetal.,2011).

OnengofEbolaZaire95(9.34×107_genome_copies)_RNA,1_ng_of

vacciniaLister(4.76×106_genome_copies)_DNA,₁_ng_of_B._anthracis

Ames(1.77×105_genome_copies)_DNA_and₁_ng_of_RVF_(1.48_×₁₀8

genomecopies)RNAweremixedandthemixturewasampliﬁed

usingthefourprotocolsrespectively.

2.4. Samplepreparationforlimitofdetectiontesting

Vaccinia Lister DNA was 10-fold serially diluted from

100,000copiesto100copiesforlimitofdetectiontestingwithout

(3)

also10-foldseriallydilutedfrom10,000copies to10copiesand

ampliﬁedusingtheRandomPCR2protocol,andtheentire

ampli-ﬁedproductwashybridizedtothearray.Copynumberquantitation

wasperformedbytheAdvancedBiotechnologiesInc.(Columbia,

MD)byrealtimePCRusingtheRocheLightCycler.

RSVwastiteredatLLNLandtissuecultureinfectiousdose50

(TCID50)wasdetermined.TheviralRNAwas10-foldseriallydiluted

from 1,000,000TCID50 to 1000TCID50 and reverse transcribed

intocDNA.Double-strandedcDNAwassynthesizedaccordingto

theInvitrogenSuperScript® _{Double-Stranded}_cDNA_Synthesis_Kit

instructions.RSVRNAwasalso10foldseriallydilutedfrom100

TCID50to0.1TCID50andampliﬁedusingRandomPCR2

ampliﬁ-cation.Theentireampliﬁedproductwaslabeledandhybridizedto

the4-plexarrays.

2.5. Samplelabelingandmicroarrayhybridization

The72K4-plexDNAmicroarraysweresynthesizedbyRoche

NimbleGen.Fourdifferentsamplescanberunoneachofthe

subar-rayssimultaneouslytosavetimeandcost.Ampliﬁedorunampliﬁed

DNAorcDNAsampleswereﬂuorescentlylabeledusingtheRoche

NimbleGenOne-ColorDNALabelingKitaccordingtothe

recom-mendedprotocols.Thelabeledsamplewaspuriﬁedafterlabeling,

andhybridizedusingtheNimbleGenHybridizationKittothe4-plex

72Karrayaccordingtomanufacturer’sinstructions.

For limit of detection of adenovirusGomen strain DNA,the

concentrationofﬂuorescentlylabeledDNAwasmeasuredwitha

Qubitﬂuorometerandthenumberofnanograms/genome

equiva-lentwasdeterminedbasedonGenBankchromosomalandplasmid

genome sizes. The inputquantity to the labelingreaction was

250ng(6.52×109_copies)._Following_labeling,_{approximately}₁₆_␮g

(4.16×1011_copies)_of_Cy3-labeled_DNA_was_obtained._The_labeled

samplewasseriallydiluted2foldfrom2␮g(5.22×1010_copies)_to

0.5ng(1.3×107_copies)_and_hybridized_to_the_arrays.

Themicroarrayswereallowedtohybridizefor 17hor1hat

42◦C, and washed using the NimbleGen Wash Buffer Kit (Cat.

05584507001) as described (Gardner et al., 2010).Microarrays

were scanned on an Axon GenePix 4000B 5␮M scanner from

MolecularDevices(Sunnyvale,CA).

2.6. Microarraydataanalysis

MicroarraydatawereanalyzedusingtheCompositeLikelihood

Maximizationmethod(CLiMax)asdescribed(Gardneretal.,2010).

Thetargetdatabaseforthe4-plex72Karraywasbuiltatalater

datethanthatusedforthe388KversionoftheLLMDA;inallother

respects,theanalysiswasthesameas reported(Gardneretal.,

2010).Probeswithsignalintensityabovethe99thpercentileof

therandomcontrolintensitieswereconsideredpositive,forthe

purposeofdataanalysis.

3. Results

3.1. Comparisonofdifferentprotocolstoamplifyamixtureof

pathogens

Fourdifferentrandomampliﬁcationprotocolswereperformed

onamixtureofbacterialandviralnucleicacids,containingDNA

orRNAfromB.anthracisAmes,RiftValleyfevervirusstrainZH501,

vacciniavirusstrainLister,andEbolavirusstrainZaire95,to

evalu-atetheefﬁcienciesofthedifferentprotocols.Allfourampliﬁcation

productswerecorrectlydetectedbymicroarraytothespecieslevel

(Table2).TheloglikelihoodisestimatedfromtheBLAST

similar-ityscoresoftheprobestoeachofthepossibletargetsequences,

togetherwiththeprobesequencecomplexityandothercovariates

derivedfromtheBLASTresults.EbolavirusZaire95wasdetected

Table2

Microarrayanalysisofamixtureofviralandbacterialtemplatesafterdifferent

randomampliﬁcationprotocols.

Ampliﬁcationmethod Detectedvirusesandbacteria Log-oddsscore RandomPCR1 Bacillusanthracis 474.5

Cowpoxvirus 216.5 Variolavirus 15.0

RVFvirus 126.2

EbolaZaire95 111.1 RandomPCR2 Bacillusanthracis 473.3 Vacciniavirus 210.3 Variolavirus 13.6

RVFvirus 124.9

EbolaZaire95 110.0 SigmaTransPlex Bacilluscereus 132.9 Vacciniavirus 223.1 Variolavirus 18.8

RVFvirus 136.6

EbolaZaire95 115.4 QiagenWTA Bacillusanthracis 504.1 Cowpoxvirus 229.8 Variolavirus 17.2

RVFvirus 137.2

EbolaZaire95 109.8

Resultsfromthemicroarraydatawereanalyzedusingthecompositelikelihood maximizationmethod.Theconditionallog-oddsscoresforthesamplesareshown. TheloglikelihoodisestimatedfromtheBLASTsimilarityscoresoftheprobestoeach ofthepossibletargetsequences,togetherwiththeprobesequencecomplexityand othercovariatesderivedfromtheBLASTresults.Adetailedlistofdetectedtarget sequencesisprovidedinSupplementaryFile1.

atthestrainlevel.Allthreesegments,S,LandMoftheRVFgenome weredetectedonarraysusingallfourmethods.RandomPCR1and 2andtheQiagenWTAkitdetectedtheB.anthracischromosome atthespecieslevel.ArrayanalysisoftheSigmaTransPlex prod-uctgaveahigherscoretoastrainofthecloselyrelatedB.cereus speciesratherthantoB.anthracis.Orthopoxviruseswerecorrectly detectedbyallfourarrays;theRandomPCR2andSigmaTransPlex arrayscorrectlyidentifiedvacciniavirusasthetopscoringspecies, whiletheRandomPCR1andQiagenWTAarraysidentifiedcowpox virusasthetophit.Allfourarraysassignedahigherexplanatory scoretoamixtureofvacciniaorcowpoxwithvariolavirusthan tovacciniaorcowpoxalone.Ofthefourprotocolstested,Random PCR2performedslightlybetterintermsofaccuracy.Inthisstudy, theRandomPCR2protocolwasusedtodeterminethedetection limitofamplifiedpathogensonthemicroarray.

3.2. Detectionsensitivityofvacciniavirus

VacciniavirusListerDNAquantitiesof100,1000,10,000and 100,000genomecopiesper12␮Lsamplevolumewereappliedto the4×72Karraytoanalyzeitslimitofdetection.Table3shows

thefractionofprobesspeciﬁctovacciniavirusListerstrainthat

had intensitiesgreaterthanthe99thpercentileof thenegative

controlsatdifferentviralconcentrations.Twoindependent

biolog-icalreplicateexperimentswereperformed.Theaverageofthetwo

replicatesresultedinmorethan70%oftheprobesbeingabovethe

detectionthresholdat100,000copiesofviralDNA,ora

concentra-tionof14fM.

VacciniavirusListerDNAquantitiesof10,100,1000and10,000

genomecopieswereampliﬁedusingtheRandomPCR2method,

and theampliﬁcation productswere hybridizedtothe4×72K

MDAarrayinordertoanalyzeitslimitofdetectionwhen

com-bined with PCR ampliﬁcation. Two replicate experiments were

performed.ThearraywasabletodetectvacciniaListerstrainwith

only100genomecopiesasinput,with72and82% of

(4)

Table3

DetectionofunampliﬁedandampliﬁedVacciniaListerviralDNAonthe72KLLMDAarray.

Vacciniavirus Nonampliﬁed Ampliﬁed

DNAcopynumberinreaction 100 1000 10,000 100,000 10 100 1000 10,000

%ofVacciniaListerstrainprobesdetectedin eachreplicate

0% 0% 0% 79% 5% 82% 100% 100%

0% 0% 20% 68% 14% 72% 100% 100%

ThesensitivityofthearrayatdifferentVacciniaListervirusDNAconcentrationswascalculatedusingthepercentageofprobesspeciﬁctoVacciniaListerstrainvirusthatwere detectedonthearray.Tworeplicateexperimentswereperformedandthepercentagedetectedoutof39VacciniaListerstrainspeciﬁcprobesisreportedforeachreplicate.

3.3. DetectionsensitivityofRSV

RSVcDNAquantitiesof1000,10,000,100,000and1,000,000 TCID50 per 12␮L sample volume were applied to the 4×72K

array.Table4shows theresponse ofthearrayat differentRSV

concentrations.Tworeplicateexperimentswereperformed.The

arraywasabletodetectRSVcDNAat100,000TCID50,withan

aver-ageof90%ofRSV-speciﬁcprobesabovethedetectionthreshold.

RSVcDNAquantitiesof0.1,1,10and100TCID50wereampliﬁed

byrandomPCRandappliedtothe4×72KMDAarray.Tworeplicate

experimentswereperformed.ThearraydetectedRSVcDNAfrom

theproductof1TCID50,withanaverageof75%ofprobesabovethe

detectionthreshold.

3.4. Detectionofadenovirusafter1hand17hhybridization

Afterlabelinghumanadenovirustype7GomenstrainDNA,

2-foldserialdilutionsofthelabeledDNAwereperformedfrom2_␮g

to0.5ngandthedilutedDNAwashybridizedtothearraysfor17h.

TheDNAconcentrationsthusrangedfrom3.9fMto15.6pM.The

responseofthearraytotheadenovirusisshowninTable5.Atall

concentrationstested,90–100%oftheprobesspeciﬁctothe

ade-novirusstraintestedwereabovethedetectionthreshold.CLiMax

analysisofthesearraysidentiﬁedthetype7Gomenstrainasthe

tophitatallconcentrationsexceptthesmallest(3.9fM),atwhich

adenovirustype3strainNHRC1276receivedthehighestscore.

To determine thespeed of detection using this microarray,

hybridizationsofadenovirustype7GomenstrainDNAwerealso

performedfor only1h. SeriallydilutedDNA quantitiesranging

from2␮g to0.5ng of DNA werehybridizedtothearray. Over

70%ofthespeciﬁcprobeswereabovethethresholdwith2ngof

DNA,correspondingto15.6fMconcentration.CLiMaxanalysis

pos-itivelyidentiﬁedtheadenovirustype7Gomenstrainwith8ngof

DNAafter1hhybridization,comparedto1ngofDNAwith17h

hybridization.

3.5. Detectionofvirusesandbacteriafromhumanfecalsamples

Four acute gastroenteritis samples received from the CDC’s

NVSNwereanalyzedonthe72K4-plexmicroarray.These

sam-pleswerepreviouslyanalyzedusingenzymeimmunoassays(EIA)

or real-time reverse transcription (RT)-PCR to determine the

presenceofalimitednumberofknownentericpathogens(Bowen,

unpublishedresults).Sample 4496was negativeby EIAor PCR

assays.Rotaviruswasdetectedinsamples4551,4912and4949

using EIA or PCR assays. Table 6 is a summary of the viruses

andbacteriadetectedonthemicroarray.Rotavirusfromsample

4551wasnotdetectedwhenalltargetsequenceswereusedfor

analysis,butitwasdetectedwhenviraltargetsequencedatabase

only wasused in theanalysis. For sample 4949, rotaviruswas

detectedinbothreplicateswhenviraltargetsequencedatabase

onlywasusedintheanalysis,howeveronlyoneoftworeplicates

detectedrotaviruswhenalltargetsequenceswereusedinthe

anal-ysis.

Themicroarrayresultsconﬁrmedtherotavirusﬁndingsfrom

thethreesamplesthatwerepreviouslytestedpositiveforrotavirus

attheCDC.Themicroarrayalsodetectedahumancalicivirusthat

wasnotpreviouslydetectedbyPCRorEIAattheCDC.Additionally,

variousbacterialpathogensincludingE.coliF11,Salmonella

enter-icaTyphi,Enterococcus faecium,Enterococcus faecalis,Bacteriodes

intestinalisweredetectedfromthefoursamples.Thesebacteria

oftencausenosocomialinfections,gastrointestinalproblemsor

uri-narytractinfections.

4. Discussion

Microarrays,alongwithPCRandDNAsequencingareeffective

methodsformicrobialdetectionanddiscoveryusingnucleicacid

samples.Whilemicroarraysarenotassensitiveorinexpensiveas

PCRassays,theycanqueryhundredsofthousandstoseveral

mil-lionregionsofDNAinparallel(dependingonplatformandformat),

comparedtoatmostafewtensofregionsinthelargestmultiplexed

PCRassays.PCRassaysaretoolimitedforbroad-spectrum

micro-bialanalysis.Severalgroupshaveappliedmicroarraytechnology

topathogen detection for clinical diagnostics, food safety

test-ing,environmentalmonitoringandbiodefense(Wangetal.,2002;

Palaciosetal.,2007;Wongetal.,2007;Palka-Santinietal.,2009; Jaingetal.,2011;McLoughlin,2011).Onemainlimitationforthe

wideruseofpathogendetectionmicroarraysisthecostoftheassay,

whichisatleastanorderofmagnitudehigherthanPCRassays.

Amultiplexedpathogendetectionmicroarraywasdevelopedand

wasamodiﬁedversionofthepreviouslyreportedLawrence

Liver-moreMicrobialDetectionArray(LLMDA)(Gardneretal.,2010)to

furtherimprovetheefﬁciency,cost-effectivenessandthroughout

ofpathogendetectionmicroarrays.

Inthisstudy,fourdifferentampliﬁcationprotocolswere

ana-lyzed: two random PCR based protocols; the Qiagen whole

transcriptionampliﬁcationprotocol,whichusesaphi29DNA

poly-merasebasedapproach;andtheSigmaTransPlexkit,whichalso

usesPCRbasedampliﬁcation.Whenfourdifferentprotocolswere

appliedtoa mixtureof fourviraland bacterialpathogens, RVF

andEbolaviruseswerecorrectlyidentiﬁedatspeciesresolution

byallprotocols,B.anthraciswascorrectlyidentiﬁedbytwo

proto-cols,Vacciniaviruswascorrectlyidentiﬁedbytwoprotocols.The

RandomPCR2ampliﬁcationprotocolcorrectlyidentiﬁedallfour

species.

Thedetectionlimitofthearraywasevaluatedbyusingserial

dilutionsofaDNAvirus,vacciniavirusLister,andanRNAvirus,

RSV,withvariousmethodsusedtoquantifytheamountsofviral

DNA or RNA. Without whole genome ampliﬁcation,the

detec-tionlimit forRSV is 100,000TCID50.Thisissimilarin rangeto

othermolecularassayssuchastherealtimePCRassay,multiplex

ligation-dependentprobeampliﬁcation(MLPA)andadual

prim-ingoligonucleotidesystem(DPO)wherethelowestconcentration

detectedforRSV was105 _TCID

50 (Bruijnesteijn vanCoppenraet

etal.,2010).ForvacciniavirusListerDNAwithoutrandom

ampli-ﬁcation,thearraydetected100,000genomecopiesorabout20pg

ofviralDNAinthe12␮Lsamplevolume,correspondingto14fM

(5)

Table4

DetectionofunampliﬁedandampliﬁedRSVcDNAonthe72KLLMDAarray.

RSV Nonampliﬁed Ampliﬁed

TCID50valuesinreaction 1000 10,000 100,000 1,000,000 0.1 1 10 100

%ofRSVprobesdetectedineachreplicate 5% 9% 91% 100% 40% 73% 100% 100%

5% 11% 89% 100% 40% 77% 100% 100%

ThesensitivityofthearraytodetectRSVviruscDNAatdifferentRSVTCID50concentrationswascalculatedusingthepercentageofprobesspeciﬁctoRSVvirusthatwere

detectedonthearray.Tworeplicateexperimentswereperformedandthepercentageof30RSVspeciﬁcprobesforeachreplicateisreported.

ThelimitofdetectionforvacciniavirusDNAwasfoundtobe 100copieswhenrandomwholegenomeampliﬁcationwasused. Thisdetection limit is similar in range toPCR based technolo-gies. A studyusingreal time PCRassays toidentifyvariants of vacciniavirusdetectedthevirusat10fg oraround 50genome copies of vaccinia DNA (Trindade et al., 2008). The difference

in sensitivity between PCR and microarrays is partially due to

the random ampliﬁcation, using random primers, instead of a

viralspeciﬁcPCRampliﬁcation.Palka-Santinietal.hasreported

that speciﬁcmultiplexed PCR ampliﬁcationprior tomicroarray

hybridizationcanincreasethedetectionlimitofpathogensbya

factorof100–1000(Palka-Santinietal.,2009).Itispossiblethat

thesensitivityofthemicroarraywillincreaseifa targeted

mul-tiplexed ampliﬁcation is applied to the samples prior to array

hybridization.

The detection limit for the RSV virus is around 1 TCID50

whencoupledwithwholegenomeampliﬁcation.Thissensitivity

issimilartomultiplexedPCRbasedLuminexassays.Agroupthat

developedaLuminexbeadassaypaneltodetect20human

respi-ratoryvirusesreportedadetectionlimitof1TCID50forRSVtypeA

and10TCID50forRSVtypeB(Mahonyetal.,2007).

Previousevaluation oftheoriginal design ofthis microarray

hasdemonstratedthatthearraydetectedvirusesfromavariety

ofhumanclinicalsamplessuchasurine,feces,serum,skinlesion,

cerebralspinalﬂuid,trachealaspirate(Erlandssonetal.,2011).In

14 outof14 clinicalsamples,theexpectedviruswasdetected:

Herpessimplexvirus1,Herpessimplexvirus2,Human

papillo-mavirus16,Humanpapillomavirus6/16/53/61,BKpolyomavirus

(BKV),JCpolyomavirus,RotavirusA,Astrovirus,Sapovirus,Dengue

1,HepatitisCvirusinduplicate,enterovirusandRSV.Thisstudy

alsodemonstratedthatthemicroarraydetectedBKpolyomavirus

positiveurinesamplescontaining≥1000copies/mL(oran

equiva-lentof5viralcopiesinaPhi29-ampliﬁcationreaction)(Erlandsson

etal.,2011).Inanotherstudy,thearraywasusedtodetect

Kaposi-sarcomaassociatedherpesvirus, orhumanherpesvirus8 from

bladdercancersamples(Paradˇziketal.,2013).Thiswastheﬁrst

indicationoftheassociationbetweenherpesvirus8withbladder

cancer, whichfurtherdemonstrated thepotentialofthe

micro-bialdetectionarraytechnologytoidentifypathogensthatmight

belinkedtocancerandotherdiseases.

Inthisstudy,theperformanceandsensitivityofthe4-plex

ver-sionoftheLawrenceLivermoreMicrobialDetectionArray(LLMDA)

onthedetectionof viruseswasevaluated usinghumanclinical

samples.IncollaborationwiththeCDC,acutegastroenteritis

sam-plesfromtheNewVaccineSurveillanceNetworkwereappliedto

the4-plexmicroarray whichidentiﬁed rotavirus,norovirusand

severalenterobacteria(E.coliF11,SalmonellaentericaTyphi,

Entero-coccusfaecium,Enterococcusfaecalis,Bacteriodesintestinalis)from

Table5

DetectionofAdenovirusDNAafter17hror1hrhybridizationonthe72KLLMDAarray.

AdenovirusDNA 0.5ng 1ng 2ng 4ng 8ng 15.6ng 125ng 1000ng

%ofadenovirusGomenstrainprobesdetectedat1h 10% 30% 80% 90% 100% 100% 100% 100% %ofadenovirusGomenstrainprobesdetectedat17h 100% 100% 100% 100% 100% 100% 100% 100% 500pgto1␮gofadenovirusGomenDNAwasanalyzedandthe%ofprobesspeciﬁctoadenoviruswascalculated.Notallconcentrationshigherthan15.6ngareshown.The percentageofprobesspeciﬁctoadenovirusGomenstrain(67totalprobes)wasusedinthecalculation.

Table6

LLMDAanalysisofhumanfecalsamplesfromacutegastroenteritiscases.Thearraydatawasanalyzedagainstbothviralandbacterialtargetsequencedatabase.Additionally, arraywasalsoanalyzedagainstviraltargetsequencedatabaseonlytoconﬁrmthevirusesdetected.Allsampleswereperformedinduplicateandallvirusesorbacterialisted weredetectedintwooutoftwosamplesexceptwhennoted.AdetailedlistoftargetsequencesdetectedislistedinSupplementaryﬁle2.

SampleID LLMDAviralresults AvgLogodds LLMDAbacterialresults AvgLogodds 4496 HumancalicivirusHu/NLV/GII/MD145-12/1987/US 207.6 Salmonellaentericasubsp.enterica

serovarTyphistr.CT18

568.3 TTvirusstrainTTVCHN1,completegenome 170.0

4551 RotavirusAstrainS2genotype G2P[4]segment2a

7.5 EscherichiacoliF11 677.1

Salmonellaentericasubsp.enterica

serovarTyphistr.CT18

79.4

Enterococcusfaecium,Glued

fragmentsofsequence630811

69.3

Stenotrophomonasmaltophilia

K279a

58.7

4912 HumanrotavirusAisolate

rj8200/04NSP4gene

151.7 EscherichiacoliF11 807.7

EnterococcusfaecalisTX0104 215.2

4949 RotavirusAstrainCC598VP7geneb _79.2 _Bacteroides

intestinalisDSM17393

262.8

a_Only_one_out_of_two_replicates_detected_rotavirus_when_analyzed_against_viral_only_target_database._Rotavirus_was_not_detected_when_analyzed_using_all_target_sequence

database.

b_Two_of_two_replicates_detected_rotavirus_A_when_analyzed_against_viral_only_target_sequences._Only_one_of_two_replicates_detected_rotavirus_when_analyzed_against_all

(6)

thesamples.TheNVSNbeganpopulation-basedactive

gastroen-teritissurveillanceamongU.S. children<3years ofagein2006

(Payneetal.,2008,2011).CurrentsurveillanceeffortsutilizePCR

andimmunoassaystodetectrotavirusandalimitednumberofviral

pathogens,butdonotprovideacomprehensiveanalysisofother

entericpathogenspresentinsamples.Thedatasuggestedthat

bac-terialandviralco-infectionsarecommoninacutegastroenteritis

patients.Thevirusconcentrationsinthesesamplesappeartobeat

alowlevelsincethehumancaliciviruswasnotdetectedbyPCRor

EIA.Rotavirussequencesfromtwosamplesweredetectableonly

whenthedatawasanalyzedagainstviralsequencesonly.Thisis

likelyduetothehigherlevelofentericbacteriaconcentrationsin

thefecalsamples.Moresensitiveviraldetectionfromhumanfecal

samplescouldpotentiallybeaccomplishedusingatargetedsample

ampliﬁcationprotocoltoselectivelyamplifyviralfamiliesof

inter-est,whichcouldprovideadvantagesoverwholegenomerandom

ampliﬁcation.

Themultiplexedpathogendetectionarrayisausefultoolfor

rapidviralandbacterialpathogendetection.Thecostfora1-plex

microarrayisinthehundredsofdollarspersample,whilethecostof

a4-plexmicroarrayisonly25%ofthe1-plexarray.Thoughthecost

isprobablynotgoingtobeatlevelscomparabletoPCR,thisassay

canreplacehundredsofPCRreactionswithjustonereaction,

there-forethe4-plexmicroarrayincreasestimeandefﬁciencyinrapid

diagnosisofclinicalinfectionsandsurveillanceofenvironmental

pathogens.

5. Conclusion

Inanefforttoimprovecost-effectiveness,speedand

through-put,amultiplexedversionoftheLawrenceLivermoreMicrobial

DetectionArraywasdevelopedandsensitivitytestingusinga

num-berofviralagents and humanclinicalsampleswasperformed.

Theseresultshavedemonstratedthatthearrayissensitivetodetect

viralinfectionsandhavethepotentialforrapidscreeningof

bac-terialandviralinfectionsorenvironmentalsamples.Multiplexed

microarrayspresentanewopportunityforhigh-throughputand

cost-effectivescreeningofthousandsofmicrobialspecies.Aswith

anytechnologybasedonnucleicaciddetection,thecapabilities

ofmicroarraysarelimitedbythegenomesequenceinformation

availableatthetimeofdesign.Furtheradvancesinarray

technol-ogysuchasautomatedarraysampleloadingandimagescanning,

fasterhybridizationtimes,andlabel-freemethodstodetect

probe-targetbindingwillbroadentheapplicationsofmicroarrayseven

further.Ultimately,microarrayscouldbeadvancedinto

point-of-caredeviceswhichdeliverresultsinlessthananhour.

Acknowledgements

ThisworkperformedundertheauspicesoftheU.S.Department

ofEnergybyLawrenceLivermoreNationalLaboratoryunder

Con-tractDE-AC52-07NA27344.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,

intheonlineversion,athttp://dx.doi.org/10.1016/j.jviromet.2014.

01.024.

References

Berthet,N.,Reinhardt,A.K.,Leclercq,I.,Ooyen,S.v.,Batéjat,C.,Dickinson,P.,

Stam-boliyska,R.,Old,I.G.,Kong,K.A.,Dacheux,L.,Bourhy,H.,Kennedy,G.C.,Korfhage,

C.,Cole,S.T.,Manuguerra,J.-C.,2008.Phi29polymerasebasedrandom

ampli-ﬁcationofviralRNAasanalternativetorandomRT-PCR.BMCMol.Biol.9, 77.

Bierbaum,S.,Königsfeld,N.,Besazza,N.,Blessing,K.,Rücker,G.,Kontny,U.,Berner,

R.,Schumacher,M.,Forster,J.,Falcone,V.,Sand,C.,Essig,A.,Huzly,D.,Rohde,

G.,Neumann-Haefelin,D.,Panning,M.,2012.Performanceofanovelmicroarray

multiplexPCRforthedetectionof23respiratorypathogens(SYMP-ARIstudy). Eur.J.Clin.Microbiol.Infect.Dis.31,2851–2861.

BruijnesteijnvanCoppenraet,L.E.S.,Swanink,C.M.A.,vanZwet,A.A.,Nijhuis,R.H.T.,

Schirm,J.,Wallinga,J.A.,Ruijs,G.J.H.M.,2010.Comparisonoftwocommercial

molecularassaysforsimultaneousdetectionofrespiratoryvirusesinclinical samplesusingtwoautomaticelectrophoresisdetectionsystems.J.Virol. Meth-ods169,188–192.

Erlandsson,L.,Rosenstierne,M.W.,McLoughlin,K.,Jaing,C.,Fomsgaard,A.,2011.

Themicrobialdetection arraycombinedwith randomPhi29-ampliﬁcation usedasadiagnostictoolforvirusdetectioninclinicalsamples.PLoSONE6, e22631.

Gardner, S., Jaing, C., McLoughlin, K., Slezak, T., 2010. A microbial

detec-tion array (MDA) for viral and bacterial detection. BMC Genomics 11, 668.

Iscove,N.N.,Barbara,M.,Gu,M.,Gibson,M.,Modi,C.,Winegarden,N.,2002.

Rep-resentationisfaithfullypreservedinglobalcDNAampliﬁedexponentiallyfrom sub-picogramquantitiesofmRNA.Nat.Biotechnol.20,940–943.

Jaing,C.,Gardner,S.,McLoughlin,K.,Thissen,J.,Slezak,T.,2011.Detectionof

adventi-tiousvirusesfrombiologicalsusingabroadspectrumMicrobialDetectionArray. PDAJ.Pharm.Sci.Technol.65,668–674.

Mahony,J.,Chong, S.,Merante, F.,Luinstra,K.,Sinha,T., Petrich,A.,Lisle, C.,

Yaghougian,S.,Janeczko,R.,2007.Developmentofarespiratoryviruspanel

testforthedetectionoftwentyhumanrespiratoryvirusesusingmultiplexPCR andaﬂuidmicrobead-basedassay.J.Clin.Microbiol.45,2965–2970.

McLoughlin,K.,2011.Microarraysforpathogendetectionandanalysis.BriefFunct.

GenomicProteomic,342–353.

Palacios,G.,Quan,P.-L.,Jabado,O.,Conlan,S.,Hirschberg,D.,Liu,Y.,2007.

Panmi-crobialoligonucleotidearrayfordiagnosisofinfectiousdiseases.Emerg.Infect. Dis.,13.

Palka-Santini,M.,Cleven,B.,Eichinger,L.,Kronke,M.,Krut,O.,2009.Largescale

mul-tiplexPCRimprovespathogendetectionbyDNAmicroarrays.BMCMicrobiol.9, 1.

Paradˇzik,M.,Buˇcevi ´c-Popovi ´c,V., ˇSitum,M.,Jaing,C.,Degoricija,M.,McLoughlin,

K.,Ismail,S.,Punda-Poli ´c,V.,Terzi ´c,J.,2013.AssociationofKaposi’s

sarcoma-associatedherpesvirus(KSHV)withbladdercancerinCroatianpatients.Tumor Biol.,1–6.

Payne,D.C., Staat,M.A., Edwards,K.M.,Szilagyi, P.G.,Gentsch,J.R., Stockman,

L.J.,Curns,A.T.,Grifﬁn,M.,Weinberg,G.A.,Hall,C.B.,Fairbrother,G.,

Alexan-der,J.,Parashar,U.D.,2008.Active,population-basedsurveillanceforsevere

rotavirus gastroenteritis in children in the United States. Pediatrics 122, 1235–1243.

Payne,D.C.,Staat,M.A.,Edwards,K.M.,Szilagyi,P.G.,Weinberg,G.A.,Hall,C.B.,

Chap-pell,J.,Curns,A.T.,Wikswo,M.,Tate,J.E.,Lopman,B.A.,Parashar,U.D.,2011.

Directandindirecteffectsofrotavirusvaccinationuponchildhood hospitaliza-tionsin3UScounties,2006–2009.Clin.Infect.Dis..

Suo,B.,He,Y.,Paoli,G.,Gehring,A.,Tu,S.-I.,Shi,X.,2010.Developmentofan

oligonucleotide-basedmicroarraytodetectmultiplefoodbornepathogens.Mol CellProbes24,77–86.

Trindade,G.d.S.,Li,Y.,Olson,V.A.,Emerson,G.,Regnery,R.L.,Fonseca,F.G.d.,Kroon,

E.G.,Damon,I.,2008.Real-timePCRassaytoidentifyvariantsofVacciniavirus:

ImplicationsforthediagnosisofbovinevacciniainBrazil.J.Virol.Methods152, 63–71.

Victoria,J.G.,Wang,C.,Jones,M.S.,Jaing,C.,McLoughlin,K.,Gardner,S.,Delwart,

E.L.,2010.Viralnucleicacidsinlive-attenuatedvaccines:detectionofminority

variantsandanadventitiousvirus.J.Virol.84,6033–6040.

Wang,D.,Coscoy,L.,Zylberberg,M.,Avila,P.C.,Boushey,H.A.,Ganem,D.,DeRisi,J.L.,

2002.Microarray-baseddetectionandgenotypingofviralpathogens.Proc.Natl.

Acad.Sci.99,15687–15692.

Wang,D.,Urisman,A.,Liu,Y.-T.,Springer,M.,Ksiazek,T.G.,Erdman,D.D.,Mardis,E.R.,

Hickenbotham,M.,Magrini,V.,Eldred,J.,Latreille,J.P.,Wilson,R.K.,Ganem,D.,

DeRisi,J.L.,2003.ViraldiscoveryandsequencerecoveryusingDNAmicroarrays.

PLoSBiol.1(2),e2,http://dx.doi.org/10.1371/journal.pbio.0000002.

Wong,C.,Heng,C.,WanYee,L.,Soh,S.,Kartasasmita,C.,Simoes,E.,Hibberd,M.,

Sung,W.-K.,Miller,L.,2007.Optimizationandclinicalvalidationofapathogen