Rev Colomb Cienc Pecu 2018; 31(4):285-294

Revista Colombiana de Ciencias Pecuarias

Original articles

285

Design and analytical validation of a duplex PCR for Ehrlichia and Rickettsia detection in ticks¤

Diseño y validación analítica de una PCR dúplex para la detección de Ehrlichia y Rickettsia en garrapatas.

Projeto e validação analítica de uma PCR duplex para detecção de Ehrlichia e Rickettsia em carrapatos

Juan C Pérez Pérez1 Myb; Carolina Montoya Ruiz1, Ing Blgo, MSc; Esteban Arroyave Sierra1, Ing Agp, MSc; Luis E Paternina2, Blgo, MSc, PhD; Juan D Rodas*, MV, MSc, PhD.

1Línea Zoonosis Emergentes y Re-emergentes, Grupo Investigaciones en Ciencias Veterinarias (Centauro), Universidad de Antioquia, Medellín, Colombia.

2Grupo de Investigaciones Biomédicas, Universidad de Sucre, Sincelejo, Colombia.

(Received: July 21, 2017; accepted: November 24, 2017)

doi: 10.17533/udea.rccp.v31n4a05

¤ Tocitethisarticle:PérezJC,MontoyaC,ArroyaveE,PaterninaLE,RodasJD.DesignandanalyticalvalidationofaduplexPCRforEhrlichiaandRickettsiadetectioninticks.RevColombCiencPecu2018;31(4):285-294.

* CorrespondingAuthor: JuanDavidRodas.LíneaZoonosisEmergentes yRe-emergentes,Grupo Investigaciones enCienciasVeterinarias (Centauro),UniversidaddeAntioquia,Calle70No.52–21,Medellín,Colombia,Tel.:(57)(4)+2196593.Fax:(57)(4)+2196525.E-mail:jdavid.rodas@udea.edu.co

Abstract

Background: Ehrlichia and Rickettsiaaretwomajorrickettsialgeneratransmittedbyticksthataffectanumberofwildanddomesticanimalspeciesandhumanpopulationsaroundtheworld.Objective:TodesignandvalidateaduplexPCRforEhrlichia and Rickettsia in ticks. Methods:Assayvalidationincludedtestingforsensitivity,specificity,reproducibility,androbustnessofthePCR.ThegroELand23srRNAgeneswereusedforEhrlichia and Rickettsia, respectively. Results:Thelimitofdetectionwasonehundredgenecopiesper50μLofreactionforEhrlichiaspp,andonegenecopyofRickettsiaper50μLofreaction.Ingeneral,theprimersofthetestonlyamplifiedinsilicothosebacterialagentsforwhichtheywereoriginallydesigned,withtheexceptionoftheprimersforRickettsia thatalsoamplifiedMethylocystissp.Thetestwasreproducible(intermediateprecision)96.7%ofthetimesforbothagents.ThetestwasrobustenoughtotolerateconcentrationchangesofallreagentswiththeexceptionofTaqDNApolymerase.Conclusions:ThevalidationresultsindicatedthatthisPCRisusefulfordetectioninbothbacterialgeneraanditisagoodcandidatefordiagnosticvalidation.

Key words: exclusiveness, groEL, inclusiveness, robustness, rRNA 23S.

Resumen

Antecedentes: Ehrlichia spp. y Rickettsia spp. son dos de los principales géneros rickettsiales transmitidos porgarrapatasque afectan a animales silvestres, domésticosyhumanos alrededordelmundo.Objetivo:

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DiseñaryvalidarunapruebaPCRdúplexparaEhrlichia y Rickettsia en garrapatas. Métodos: La validación delapruebaincluyóensayosdesensibilidad,especificidad,reproducibilidadyrobustez.EnlaPCRseusógroEL y ARNr 23S como genes blanco para Ehrlichia y Rickettsia, respectivamente. Resultados: El límite de detecciónfuede100copiasdelgenpor50μLdereacciónpara Ehrlichia spp y una copia del gen de Rickettsia por50μLdereacción.Engeneral,loscebadoresdelapruebasoloamplificaroninsilicolosagentesbacterianosparaloscualesfueronoriginalmentediseñados,conlaexcepcióndeloscebadoresdeRickettsia que también amplificaronMethylocystissp.Lapruebafuereproducible(precisiónintermedia)enun96.7%delasvecesparaambosagentes.Lapruebafuesuficientementerobustacomoparatolerarcambiosdeconcentracióndelosdiferentesreactivos,conexcepcióndelaTaqDNApolimerasa.Conclusión: Los resultados de validación indican que la PCR es útil para detectar ambos géneros bacterianos y podria usarse para validación diagnostica.

Palabras clave: ARNr 23S, exclusividad, groEL, inclusividad, robustez.

Resumo

Antecedentes: Ehrlichia e Rickettsia são dois dos principais gêneros de rickettsias transmitidos por carrapatosqueinfectamtantoanimaisselvagensquantoanimaisdomésticoseatéhomensemtodoomundo.Objetivo:OobjetivoprincipalfoielaborarevalidarumaPCRduplexparaEhrlichia e Rickettsia em carrapatos. Métodos:Avalidaçãoincluiu testesdesensibilidade,especificidade, reproduçãoerobustez.ParaoPCR,utilizamososgenesgroEle23Sr-RNAparaEhrlichia e Rickettsia, respectivamente. Resultados:Olimitededetecçãofoide100cópiasdegenespor50mldereaçãoparaErliquia spp e uma cópia de gene de Rickettsia por50mldereação.Emgeral,osiniciadoresdostestesamplificaramemmodeloscomputacionaisosagentesbacterianospara os quais eles foramprojetados, excetoos primers deRickettsia que tambémamplificouMethylocystissp.Ostestesforamreproduzíveis(precisãointermediária)96,7%paraambososagenteseforamtambémrobustosparatolerarmudançasdeconcentraçãoemtodososreagentes,excetooreagenteTaqDNApolymerase. Conclusões: Os resultados da validação indicaram que o PCR é útil para detecção em ambos os gênerosbacterianos,portanto,umbomexameparavalidaçãodiagnóstica.

Palavras-chave: exclusividade, groEL, inclusividade, robustez, rRNA 23S.

Introduction

Ticksareobligateectoparasitesofmanyvertebrateclassesaround theworld.Aftermosquitoes, ticksareconsideredthesecondmostimportantvectorforhumandiseases (Pfäffleet al., 2013). In spiteof thebroaddiversityoftickspecies,themajorityofthembelongtothe genera Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, and Rhipicephalus,intheIxodidaefamily(Socolovschiet al.,2009).

Many agents transmitted by ticks are considered zoonotic. Among them it is worth mentioning the alpha-proteobacterialagents Ehrlichia and Rickettsia (Dantas-Torreset al., 2012; Moriello, 2007). Both areintracellularobligategram-negativebacteriaandaffectagreatvarietyofvertebratehosts, includinghumans and dogs (Mansueto et al., 2012; Rar and Golovljova,2011).

The Rickettsiagenusaredividedintofourmajorgroups,knownasSpottedFever(SFG),Typhus(TG),Ancestral (AG), andTransitional group (TranG)

(Quintero Vélez et al., 2012; Mansueto et al., 2012). On the other hand, the Ehrlichia genus includes E. chaffeensis, E. canis, E. ewingii, E. ruminantium, E. minasensis, and E. muris (Rar andGolovljova,2011,Cabezas-cruzet al., 2016).RockyMountainSpottedFever(RMSF),causedbyRickettsia rickettsii, remains as the most deadly rickettsial human disease (Hidalgo et al. 2007, Quintero Vélez et al., 2012), while Canine Ehrlichioses (CE) caused by Ehrlichia canis is the most important and widespread rickettsial disease among dogs (Moriello et al, 2007).

ThePCR-basedmethodsarethebestchoicefordetecting both rickettsial agents in ticks due to its high sensitivity and specificity (Oteoet al., 2014). However, Ehrlichia and Rickettsia detection in infectedticksremainsasthemostchallengingtaskinecologicalstudiesoftick-borneagents.Somegenes,suchas16SribosomalRNA,dsb, p30 and p28, are commonlyusedformoleculardetectionofEhrlichia (Doyle et al., 2005),whilegltA, ompA and ompB genesareusedfordetectionofRickettsia (Oteo et al., 2014; Quintero Vélez et al.,2012).Theidentification

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ofrickettsialagentsisadifficultprocessthatusuallyrequiressequencingandphylogeneticanalysisofoneor several bacterial genes.

Despite Ehrlichia and Rickettsiabacteriaeco-infectthesameticks(Bowman,2011;Dantas-Torreset al., 2012;DumlerandWalker,2001;RarandGolovljova,2011)andthenumerousPCRstrategiesavailablefordetectionofrickettsialagents in ticks, therearenoreports of PCRmethodologies for simultaneouslydetecting both Rickettsia and Ehrlichia in ticks. Basedontheaforementionedarguments,theaimofthisworkwastodevelopandvalidateaduplexPCRfor detection ofEhrlichia and Rickettsia in DNA extractedfromticks.

Materials and methods

Rickettsial genes and oligonucleotides design

We retrieved sequences ofEhrlichia groESL gene and Rickettsia23Sribosomalgenefromgeneticdatabases (GenBank/EMBL/DDBJ) and alignedthe DNA sequences using the ClustalW algorithm (Thompsonet al.,1994)toidentifyconservedregionsforprimerdesign.ThePrimer-BLASTtoolofNCBI(forkofPrimer3software)wasusedforthedesignandpreliminaryevaluationoftarget-specificityofprimers(Yeet al., 2012.). We calculated oligonucleotide properties suchasdimerizationpotential,hairpinformationandTm inPerlPrimer1.1.21 software (Marshall,2004).Primers selected forEhrlichiawereEhr-GroELF5´CAATAGCAAGAGCCAATG3´ andEhr-GroELR5´TTAGAAGATGCTGTAGGATG3´andamplifiedtoasizeof145bp and RickettsiawasRick-23SF5´CAGACTTACCAAACTCAATC3´andRick-23SR5´TACGCAAGAACCCTTGGA3´amplifiedto 437 bp.

Ticks naturally infected with Ehrlichia and Rickettsia

As positive controls forEhrlichia-Rickettsia duplex PCR assays,we isolatedDNA from tickspositiveforEhrlichia canis dsb gene by PCR, and other ticks positive to gltA and ompB genes ofRickettsia sp.AtlanticRainforest, these tickswerestored at the laboratory of theCentauro researchgroup (Universidad deAntioquia).DNAof ticks

wasextractedfromindividualcompleteticksusingaQIAGENDNeasyBloodandTissuekitaccordingtomanufacturer instructions, and itwasquantifiedby nanodrop.DNase andRNases freewaterwasusedasanegativecontrolandGenomicDNAofR. rhipicephali, R. amblyommatis, R. parkeri and R. bellii, kindly provided by Dr. Marcelo B. Labruna from UniversityofSãoPaulo(Brazil),werealsousedas positive controls.

Ehrlichia and Rickettsia duplex PCR (ER-dPCR)

PCR reactionswere carried out in aGeneAmpPCR System 2400 thermocycler (Applied Biosystems, FosterCity,CA,USA) in aPCRvolumeof 50μLwith aPCRcocktailmixture as shown inTable1.Amplification conditionswere 95 °C for 1min,followed of 40 cycles to 95 °C/30 s, 56 °C/30 s,72°C/30sandafinalextensionstep72°C/7min.Electrophoresiswasperformedin2%AgarosegelinTAE1Xusing0,25μLofGelRed(Biotium)foreach10mLofgel.Electrophoresiswasrunwith100Voltsduring45minutes.GeneRuler100bpPlus(ThermoScientific)was used asmolecularweightmarker.Photodocumentationofgelswasperformedwith aDoc™XR+Systeminstrument(Bio-Rad).

Table 1. PCR cocktail for ER-dPCR.

Reagent [Initial] [Final] Volume (µL)

Nuclease free water NA NA 30.75

PCR Buffer * 10 X 1 X 5

dNTPs** 10 mM 0.2 mM 1

Ehr-groELF *** 10 µM 0.2 µM 1

Ehr-groELR *** 10 µM 0.2 µM 1

Rick-23SF *** 10 µM 0.2 µM 1

Rick-23SR*** 10 µM 0.2 µM 1

MgCl2 * 25 mM 3 mM 6

Taq DNA polymerase * 5 U/µL 1.25 U 0.25

Tick DNA 100 ng/µL 300 ng 3

* Thermo Scientific; ** Invitrogen; *** Bioneer.

Detection threshold of ER-dPCR (sensitivity)

groESL amplicon of Ehrlichia canis and 23S amplicon of Rickettsia sp.AtlanticRainforestwasinserted into pDrive Cloning Vector (QIAgen). Concentrationof theplasmidswas adjusted to108

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plasmidsorgenecopies/μLinten-foldserialdilutionstoobtainasinglecopyofeachgene/μL.Threereplicasper dilution were tested in each PCR to determine the minimumnumberofcopiesofeachrickettsialgeneamplifiablebyapositivePCR.

Robustness assay for ER-dPCR

TheperformanceoftheduplexPCRwasassayedunder three stepwise concentrationsofMgCl2 (2.5mM;3.0mM;3.5mM),dNTPs(0.1mM;0.2mM;0.3mM),TaqDNApolymerase(1.0U;1.25U;1.5U), oligonucleotide concentration (0.2 µM; 0.4 µM; 0.6µM),variationintemperaturealignment(54,56,58°C),andwealsotestedthreedifferentbrandsofTaqDNAPolymerase(ThermoScientific,InvitrogenandBioline).Threereplicasforeachreagentconcentrationweretestedinallexperiments.

Repeatability and reproducibility for ER-dPCR

Repeatability was assayed by testing the same DNA sample (tick DNA with three replicas) in the ER-dPCRbythesameperson,alongfiveconsecutivedays in the samePCRmachine.The same analystperformed all PCR in the same thermocycler.Reproducibility(intermediateprecision)oftheER-dPCRwastestedbymakingthesameexperimentsoftherepeatabilityassay,butperformedbyadifferentperson.The results of successful ER-dPCRwereexpressedaspercentagesinbothassays.

Inclusiveness and exclusiveness assay of ER-dPCR

ThePrimer-BLASTandInSilicoplatforms(http://insilico.ehu.eus)wereusedtodeterminespecificityofEhrlichia and Rickettsia primers. During the evaluationofeacholigonucleotide,R. amblyommatis, andDNAofotheragentstransmittedbyticks-suchas Babesia and Borrelia- were also tested in order to excludeunspecificamplification.

Phylogenetic trees. phylogeneticanalysisof thesequenceswith the amplicons downloaded fromthesimulationofInsilicoplatformswascarriedoutforwhich thebestmodelwasdetermined, and theconstruction of the treeswas performed byMega6.06usingthemethodsofmaximumlikelihoodandneighborjoiningwitha1000Bootstrapreplications.

Results

Detection threshold of ER-dPCR

ThethresholdfordetectionofEhrlichia was 100 copiesofgroESLgeneper50μLofreaction(2genecopies per μL), and the threshold for detectionofRickettsia was1copyof23Sgeneper50μLofreaction(0.02genecopiesperμL)(Figure1).

Figure 1. DetectionthresholdassayforER-dPCR.R-Rickettsia (437bp),E-Ehrlichia (145bp).

Robustness of ER-dPCR

Theduplexmethodologywasrobustenoughtoallowthesimultaneousamplificationofbothrickettsialagentsinall theevaluatedconcentration rangesof reagents,exceptfortheminimumvalueofTaqDNAPolymerase(1U)whereno amplificationof any fragmentwasobserved. In addition, theER-dPCR successfullyamplified both rickettsial agents regardless ofTaqpolymerasebrandused(ThermoScientific,BiolaseandInvitrogen).Thismolecularprocedurewasalsorobustforalignmenttemperaturechangesbetween54to58°C.ResultsoftherobustnessassayareshowninFigure2.

Repeatability and reproducibility

Ehrlichia testing repeatability of analyst #1was100%(15/15),while forRickettsia testing the repeatabilitywas93.3%(14/15).Ontheotherhand,

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therepeatabilityofanalyst#2was93.3%forEhrlichia detection(14/15)andrepeatabilityforRickettsia was 100% (15/15).The reproducibility (intermediateprecision) for both agentswas 96.7% (29/30), thesummaryofbothtestsispresentedinTable2.

Inclusiveness and exclusiveness assays. ThePrimer-BLAST tool ofBCBI shows that primersEhr-groELF/Ehr-groELRcanamplify41sequencesofdifferentspeciesofEhrlichia genus (Supplementary file1),andtheseoligonucleotidesdonotshowcross-amplificationwithorganismsotherthanEhrlichia. E. canisinfectedtickswereanalyzeduponavailabilityinourlaboratory(seefigure3A).

Figure 2. RobustnesstestofER-dPCR.R-Band437pbofRickettsiaspp.E-band145pbofEhrlichiaspp.R1-replica1.R2-replica2.R3-replica3.R0-negativecontrol.

Table 2. Repeatability and reproducibility results.

Analyst # Day 1 Day 2 Day 3 Day 4 Day 5 Repeatability Reproducibility

Ehrlichia 1 3/3 3/3 3/3 3/3 3/3 100% (15/15) 96.6% (29/30)

2 3/3 3/3 3/3 2/3 3/3 93.3% (14/15)

Rickettsia 1 3/3 2/3 3/3 3/3 3/3 93.3% (14/15) 96.6% (29/30)

2 3/3 3/3 3/3 3/3 3/3 100% (15/15)

ThePrimer-BLASTanalysisofRick-23SF/Rick-23SRshowsthattheycanamplify79sequencesofdifferentRickettsia species (Supplementaryfile 3),potential cross-amplificationwas observed for onenon-rickettsial target:Methylocystis sp. SC2 strain SC223SribosomalRNA(NR_077052,437pb).Thepotentialcross-amplificationofMethylocystis could be considered as irrelevant since this bacterium is a free-livingmicroorganism so far not found inticks. In vitro testingof theER-dPCRwas able toamplifyR. rickettsii, R. rhipicephali, Rickettsia sp AtlanticRainforest, R. parkeri, R. amblyommatisi, R. felis, and R. bellii DNA(seefigure3A). TheInSilicotestingshowssuccessfulamplificationforall

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Rickettsiaspecies(41referencesequencesavailableintheplatform),nocross-amplificationwasobservedforAnaplasma, Ehrlichia, Wolbachia, Borrelia and Neorickettsia (Supplementary file 4). Further in vitroevaluationshowsnocrossamplificationofER-dPCR in Wolbachia, Babesia bigemina and Borrelia anserina (seeFigure3B).

AdendrogramofEhrlichia genus using partial sequencesofgroELandNeighbor-JoiningalgorithmretrievedfromInSilicoplatformshowsthatEhrlichia speciescanberapidlydistinguishedinspiteofthesmallfragmentanalyzed(Figure4A).Fromthedendrogram,using the 23S Rickettsia partial sequences, it is obvious that species discrimination inside theSFGR is notpossible (red circle), although discrimination inside the TGmightbepossible(Figure4B).

Discussion

Currently,thereisalargenumberofPCRstrategiesfor detection ofRickettsia and Ehrlichia. Thesetechniques are good for individual ormultiplexedamplification with other tick-borne agents inconventional (Corales et al., 2014; Jado et al.,2006;Kledmanee et al.,2009;Levineetal,2010;Regneryet al.,1991;Rufinoet al., 2013; Stevenson et al, 2003; Sumner et al., 1997;Warner et al., 1997), nested(Massung et al,, 1998), and real-timePCR (Doyleet al., 2005;Killmaster et al., 2014;Loftiset al., 2006a,2006b,2003;Pariset al., 2008; Peleg et al., 2010; Quarsten et al.,2015).Amongthose,real-timePCRstrategiesrepresentthenewstandardforhighlysensitiveandspecificdetectionofRickettsialagentsin ticks; however, conventional PCR still remains as themostcommonmolecularplatformforselectiveDNAamplification.

ThemostcommonlyusedgenefordetectionofAnaplasmatacea agents (Anaplasma and Ehrlichia) is16SrRNA(Killmasteret al., 2014; Levine et al., 2010;Loftiset al.,2006b;Massunget al.,1998;Peleget al.,2010;Rufinoet al., 2013; Warner et al.,1997),whereas dsb gene is the standard for identificationof Ehrlichial agents (Doyle et al., 2005).On theother hand, groEL andvirB9genesareconsideredas alternative genes forEhrlichia identificationthrough genetic analysis (Levine et al., 2010; Sumner et al.,1997;Coraleset al., 2014; Kledmanee et al., 2009).OnRickettsia detection, the citrate synthase gene(gltA),hasbeendeemedasagoldstandardforlong time (Paris et al., 2008; Quarsten et al.,2015;Regnery et al.,1991;Rouxet al.,1997;Oteoet al., 2014), but other genes such as 17KDa genus common rickettsial antigen and23S-5S internal transcribedspacerhavealsobeenusedandtestedforthesametask

Figure 3. Inclusivenessandexclusiviness in vitro tests.R-band437pbofRickettsiaspp.E-band145pbofEhrlichia spp.C+PositivecontrolwithRickettsia spp. and Ehrlichia spp.C-Negativecontrol(waterfreeofDNasesandRNases).A.Inclusivenesstest.B.Exclusivinesstest.R1Replica1.R2Replica 2. R3 Replica.

In the InSilicoplatform, theEhrlichia primers didnotallowthesuccessfulamplificationofgroESLgene when genomes ofRickettsia, Wolbachia, Borrelia, Anaplasma and Neorickettsia were used (Supplementaryfile 2).The InSilico test ofEhrlichia primers against complete genomes ofE. canis str. Jake, E. chaffensis str. Arkansas, E. muris AS145,E. ruminantiumstr.Gardel,E. ruminantium str. Welgevonden CIRAD and E. ruminantium str. Welgevondenproducesampliconsofexpectedsize.ThesequencesofEhrlichialamplicons(andE. ewingii AF195273)wereusedtobuildaphylogenetictreeofgroELgeneasshowntheFigure4A.

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Figure 4. Phylogenetic trees. A. Phylogenetic tree Ehrlichiaspp.TreewasperformedusingthedownloadedsequencesoftheapplicationINSILICOPCR.TreeperformedbyneighborjoiningusingtheTamura1992modelhasinvariantsites(I),witha1000 Bootstrap replications. B. Phylogenetic tree Rickettsiaspp.TreewasperformedusingthedownloadedsequencesoftheINSILICOPCRapplication.ThetreeperformedbymaximumlikelihoodusingKimura2modelparameterswithgammadistribution(G),witha1000Bootstrapreplications.Circle-Spottedfevergroup.Square-AncestralGroupTriangle-Transitionalgroup.Diamond-TyphusGroup.

B

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(Killmaster et al.,2014;Loftiset al.,2006a;Stevensonet al., 2003; Jado et al.,2006).Inthepresentstudy,we developed a sensitive conventional PCR strategy forsimultaneousandspecificdetectionofEhrlichia and Rickettsia in ticks.

Co-infectionwithmultiplerickettsialagentsisacommon scenario in ticks. Actually, there are reports of simultaneous infection byRickettsia, Ehrlichia chaffensis and Ehrlichia ewingii in Amblyomma americanum in Georgia (USA) (Killmaster et al., 2014), and a more recent report describes the occurrence of common bacterial co-infections inIxodes ricinus inFrance (Moutailleret al., 2016).Themoleculardetectionofco-infectionbyseveralbacterial agentswould requireperforminga singlePCRforeachconsideredagent,ordevelopingnewmultiplexandduplexPCRsassays.

TheER-dPCRhasshownpromisingresultswithadetectionthresholdforRickettsia about0.02copies/μLand2copies/μLfordetectionofEhrlichia. Our resultsforRickettsia detection threshold are similar tothePCR-RLBdesignedbyJadoet al.,2006thatdetectsupto0.02copies/μL,andbetterthantheoneobtained by Paris et al., (2008) that reports detection upto1copy/uLforTGRand2ompBgenecopies/uLforSFGRinamultiplexedSYBR-qPCRassay.Ontheotherhand,thedetectionthresholdofER-dPCRforEhrlichia(2copies/uL)issimilartoTaqman-qPCRreported by Doyle et al.(2005),thatdetects1copy/μLofanyEhrlichia.

Duplexed detection techniques have beendeveloped forE. canis and the protozoa B. canis vogeliwiththresholddetectionbetween1-10genecopies (Peleg et al., 2010). Other conventional PCR assays targeting the groEL region claim to detect2-genecopies/μL(Levineet al., 2010), and in our case the Ehrlichia detection showed similar sensitivity to the aforementioned techniques.Moreover, other strategies focused on qPCR andconventional PCR reported a threshold detection forRickettsiaabout2-genecopies/μL(Killmasteret al.,2014;Loftiset al.,2006);accordingtothis,ourER-dPCRiscomparabletootherstrategiesavailablein the scientific literature,with the advantage thatthisonedoesnotlosesensitivityindetectionofbothrickettsial agents.

Oneof themajorconcernsregardingtheuseofduplexedormultiplexedPCR techniqueshasbeenthepoorreproducibilityandinstabilityoftheassays.However, as we showed in the repeatability and reproducibilitytests(intermediateprecision),theER-dPCR is accurate enough to ensure its use in research, haspromisingfeaturesforitsuseinclinicaldiagnosis,and itseemsnot tobegreatlyaffectedbydifferentusers.WealsoshowthatER-dPCRishighlyrobustand tolerateswide rangesof reagent concentrationandcommercialbrands,withtheonlyexceptionthatitrequiresaminimalamountofTaqDNApolymerase(1,25Uperreaction)forgoodperformance.

Theinclusivenessandexclusivenesstest(insilicoand in vitro)showedthatthedesignoftheER-dPCRis specific forEhrlichia and Rickettsia bacterial generaanddoesnotamplifyothermicroorganismscommonlyfoundinticksaroundtheworld,suchasAnaplasma, Borrelia, Babesia or Wolbachia.Thephylogeneticsignalof the23SregionofRickettsia used in our study is good enough to differentiateamong the main bacterial groups within this genus. However, it is not suitable to differentiate amongSFGRspecies,whereasdifferentiationofEhrlichia species using a small groEL region is possible, as demonstrated in previous studies (Sumner et al., 1997).AbiglimitationofourworkwastheinabilitytotestforotherEhrlichia agents, since other species ofthisbacterialgenusarefoundintheneoarcticandpaleartica regions and only E. canisiswidelyfoundacrosstheworld.TheER-dPCRdescribedherewasdesigned forEhrlichia and Rickettsia detection in ticks,butitcanalsobeappliedtothestudyofdogpopulations,sincenoneofthetargetsshowspotentialcross-amplificationwithcarnivores.Finally,furthervalidationofourrickettsialdetectionstrategymustbe conducted to establish the epidemiological utility oftheER-dPCRforrickettsialdiseases.

Inconclusion,wedevelopedarobustduplexPCRforthereliabledetectionofEhrlichia and Rickettsia agents,comparabletootherhigh-performancesingle-detectionstrategies.Tothebestofourknowledge,thisisthefirstrickettsialPCRperformedwithanalyticalvalidation.

Furthervalidationofthetestisplannedusingfieldcollectedticksandticksfromdogs.

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Acknowledgments

This study was funded by COLCIENCIAS,Colombia(call585,year2013).

Conflicts of interest

The authors declare they have no conflicts ofinterest with regard to the work presented in this report.

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