Improved detectionby RT-PCR of heat shock-inducible hsp70 gene(1).pdf

Research BriefGiardia duodenalis:Improved detection of viable cysts by reversetranscription-PCR of heat shock-inducible hsp70 geneGyu-Cheol Leea,*,Se-Hee Nama,Jong-Chan Chaeb,Chan-Hee LeecaWater Analysis and Research Center,K-Water,Daejeon 306-711,Republic of KoreabDivision of Biotechnology,College of Environmental and Bioresource Sciences,Chonbuk National University,Iksan 570-752,Republic of KoreacSchool of Life Sciences,College of Natural Sciences,Chungbuk National University,Cheongju 361-763,Republic of Koreaa r t i c l ei n f oArticle history:Received 12 March 2009Received in revised form 14 August 2009Accepted 18 August 2009Available online 22 August 2009Keywords:Giardia duodenalisRT-PCRhsp70Heat shocka b s t r a c tGiardia duodenalis is a waterborne protozoan parasite that causes the diarrhoeal disease,giardiasis.Itsdurable and thick cell wall allows the parasite to exhibit resistance to environmental stresses.BecauseG.duodenalis exists in a water system at low levels,it is necessary to develop a sensitive method to detectits viability in aquatic environments.In the present study,specific primers for the heat shock protein(hsp)70 gene were designed on the basis of G.duodenalis genome sequence and bioinformatic analysis.Viable G.duodenalis cysts were successfully distinguished by reverse transcription-PCR(RT-PCR)analysisusing these primers.The amplicon of hsp70 was obtained from one cyst of G.duodenalis/100ll,and thisdetection sensitivity significantly increased by 103-fold when the cysts were given heat shock treatment.These findings prove that viable G.duodenalis cysts were successfully detected with a high degree of sen-sitivity by RT-PCR analysis targeting the hsp70 gene of G.duodenalis,thereby suggesting its practicalpotential for detecting viable G.duodenalis in environmental samples.?2009 Elsevier Inc.All rights reserved.1.IntroductionIt has been well established that Giardia duodenalisa water-borne protozoan parasitecauses giardiasis in association withCryptosporidium parvum(C.parvum),which causes cryptosporidi-osis(Adam,2001;Berkman et al.,2002).G.duodenalis causes gas-troenteritis,such as nonbacterial diarrhoea,and is released intorivers or lakes via the faeces of its hosts such as human andmammals(Adam,1991,2001;Marshall et al.,1997).The contam-inated water,therefore,is considered a source of the diseasecaused by G.duodenalis(Fricker et al.,2002;Karanis,2006;Mah-bubani et al.,1998).G.duodenalis cysts are composed of a hard and thick cell wallthat provides resistance against various stresses.Thus,its viabilityin the environment is sustained for a relatively long period of timeand it is moderately resistant to the chlorination process in watertreatment plants(Leahy et al.,1987).Humans are easily suscepti-ble to giardiasis caused by even small numbers of G.duodenalis,and hence,it is critical to develop highly sensitive detection meth-ods and survey the incidence of G.duodenalis.In order to providesafe tap water,studies on the development of novel detectionmethods as well as studies that will provide information on theinactivation and removal of G.duodenalis are warranted.The con-ventional method used to detect G.duodenalis cysts in aquaticenvironments is that established by the U.S.Environmental Protec-tion Agency(USEPA Method 1623,2001).By using the method,thecysts of G.duodenalis are selectively separated using an antibodyspecific for G.duodenalis and the number of the cysts are thencounted using a fluorescence microscope.However,this procedurerequires several different assay steps,and can not ascertain the via-bility of G.duodenalis cysts.Further,fluctuations in the test resultscould be attributable to the difference in the skills of theinvestigator.As opposed to conventional methods,several molecular-basedtechniques such as polymerase chain reaction(PCR)are being usedto conduct highly specific and highly sensitive analysis of environ-mental water samples(Cacci,2003;Haque et al.,2007;Miller andSterling,2007).Reverse transcription(RT)-PCR by using primers fordetecting the heat shock protein(hsp)gene has been proposed as acandidate method for detecting viable G.duodenalis cysts adminis-tered artificial heat shock treatment(Abbaszadegan et al.,1997).Heat shock treatment induces the mRNA transcription of the hspgene(Abbaszadegan et al.,1997)the amplification of which wasnot only successful in this study but it was also reported that therewas little in reproducibility(Kaucner and Stinear,1998).Thus,inthe present study,we established a new RT-PCR for the hsp70 geneof G.duodenalis to detect the viable G.duodenalis cysts.0014-4894/$-see front matter?2009 Elsevier Inc.All rights reserved.doi:10.1016/j.exppara.2009.08.011*Corresponding author.Address:Water Analysis and Research Center,K-Water,560 Sintanjin-Ro,Daedeok-Gu,Daejeon 306-711,Republic of Korea.Fax:+82 42629 2079.E-mail address:gcleekwater.or.kr(G.-C.Lee).Experimental Parasitology 123(2009)377380Contents lists available at ScienceDirectExperimental Parasitologyjournal homepage: and methods2.1.Preparation and observation of the cyst of G.duodenalisThe cysts of G.duodenalis were purchased from Waterborne Inc.(New Orleans,LA,USA).They were either used immediately inexperiments or stored at 48?C and used within 14 days.The cystsof G.duodenalis were heat-killed by treating at 150?C for 15 min(Thiriat et al.,1998).Identification of the cyst of G.duodenalis was performed as fol-lows.Samples including cysts of G.duodenalis were dried on a wellslide and fixed using 100%methyl alcohol.An immunofluorescenceassay kit(Aqua-glo G/C,Waterborne Inc.)using a 40,6-diamidino-2-phenylindole(DAPI,Sigma Chemical Co.,St.Louis,MO,USA)wasused to stain the nucleus of the sample by a fluorescence dyeingprocess.The cyst of G.duodenalis was verified through the immu-nofluorescenceassay(exciterfilter,450490 nm;dichroicbeam-splitting mirror,510 nm;barrier or suppression filter,515520 nm),nucleus dyeing test(exciter filter,340380 nm;di-chroic beam-splitting mirror,400 nm;barrier or suppression filter,420 nm),and differential interference contrast(DIC)observationusing a fluorescence microscope(Eclipse E800,Nikon,Japan)withat least 400?magnifications.2.2.RNA extractionA Dynabeads mRNA DIRECTTMkit(Dynal Biotech,Oslo,Norway)was used to extract the mRNA of G.duodenalis cysts.Using 10-foldserial dilutions,the cyst of G.duodenalis were prepared as a finalconcentration of 100104cysts/100ll of phosphate buffered salinesolution(PBS).The total number of cysts at each dilution wascounted three times to confirm that the dilution steps were con-ducted accurately.When necessary,a heat shock treatment wasapplied at 42?C for 20 min to induce hsp70 mRNA transcription.After 200ll of lysis/binding buffer solution(Dynal Biotech)wasadded into each cyst suspensions,they were frozen in liquid nitro-gen for 1 min and then thawed in a water bath at 65?C for 1 min.This process was repeated six times.Thereafter,20ll of Dynabeadsoligo(dT)25-bead(Dynal Biotech),was added and incubated at30?C for 30 min in a mixer(Sample Mixer,MXICI model,DynalBiotech)and magnetic particles bound with nucleic acid were sep-arated using a magnetic particle concentrator.Next,it was washedwith the washing buffer A and B provided in the kit.Following thewashing step,the mRNA was separated from the magnetic body byheating the solution at 80?C for 2 min with 50ll of ice-cold10 mM TrisHCl solution.This was then used as a template for aRT reaction in order to produce cDNA;DNA was completely re-moved by treating the solution containing separated mRNA with0.01 U/ll DNase I(ABgene,Epsom,UK).In order to confirm geno-mic DNA contamination,PCR was performed with the extractedmRNA and using the designed primer set for the hsp70 gene.ThePCR condition was the same as mentioned later without the re-verse transcription step.2.3.RT-PCRThe RT reaction was performed using the Sensiscript RT kit(Qiagen,Hilden,Germany).After mixing 10ll of the mRNA tem-plate with 2ll of 10?RT buffer,2ll of dNTP mixture,1ll ofreverse transcriptase,2ll of PCR primers 10 pmol/ll(forward,50-GTTTAAGAAGAAGCACGGGA-30;reverse,50-GAAGTCTATGCCCTCGAAAA-30),and 1ll of 10 U RNase inhibitor,the volume of the finalsolution was adjusted to 20ll using RNase-free water(Qiagen).After RT reaction at 37?C for 60 min,a PCR was conducted usingthe synthesized cDNA.The final volume was adjusted to 20ll bymixing3llofthehsp70cDNA,2llofeachPCRprimer(10 pmol/ll),10ll of 2?Taq DNA polymerase premixture(TaqDNA polymerase 0.1 U/ll,dATP,dGTP,dCTP,and dTTP for eachof 0.5 mM,and 4 mM MgCl2)and 3ll of nuclease free water(Ambion,Austin,TX).The cycling conditions were at 94?C for5 min,followed by 40 cycles of denaturation at 94?C for 1 min,annealing at 51?C for 1 min,and extension at 72?C for 1 min,and followed by a reaction at 72?C for 7 min.The PCR reactionwas conducted using the GeneAmp PCR System 9700(Applied Bio-systems,Foster city,CA).2.4.Phylogenetic analysisPhylogenetic analysis of hsp gene(GenBank accession No.X16738)was conducted using homologues;the nucleotide se-quences of hsp70 genes(GenBank accession Nos.AB056660,AB062115,AF182195,AY029210,NM_002155,U04874,U69698,XM_001225917,XM_001707918,and XM_651738)and dyneinheavy chain genes(GenBank accession Nos.XM_001707201,XM_627924,XM_661002,XM_799723,and XM_842063)from var-ious species.Those nucleotide sequences were aligned using Clu-stalW(version 1.82 at the European Bioinformatics Institute,http:/www.ebi.ac.uk)and ClustalX(version 1.83)and a Neigh-bour-joining(NJ)tree was constructed using ClustalX program.The phylogenetic tree was viewed using TreeView program(ver-sion 1.6.6).3.Results and discussionIt has been of interest to detect G.duodenalis,in particular theviability of G.duodenalis cysts,in a water system in which it existsat very low concentration levels.To determine the viability ofG.duodenalis cysts,it is necessary to conduct experiments usinganimal models for infectivity(Connelly et al.,2007;Labatiuket al.,1991),in vitro excystation(Boucher and Gillin,1990;Riceand Schaefer,1981),or dye exclusion assays using fluorescein diac-etate,propidium iodide(PI),etc.(Schupp and Erlandsen,1987;Smith and Smith,1989).Although a PCR method for the b-giardingene of G.duodenalis has been reported,it is not considered to besuitable for verifying the viability of the detected G.duodenalis(Guy et al.,2003).To overcome these obstacles,RT-PCR was devel-oped to detect the mRNA of the hsp gene transcribed from viable G.duodenalis and induced by heat shock(Abbaszadegan et al.,1997).In order to estimate the detection of viable G.duodenalis,the RT-PCR protocol used by Abbaszadegan et al.(1997)was utilized todetect RNA from source water samples inoculated with viable G.duodenalis cysts.However,despite applying heat shock treatment,the assay had significantly low detection sensitivity(103cysts/100ll),alike the report by Kaucner and Stinear(1998)describingunreliability of the RT-PCR.As a consequence of sequencing faint RT-PCR products,thenucleotide sequences did not precisely match with that of thehsp gene(GenBank accession No.X16738,Giardia lamblia heatshock protein gene)(Abbaszadegan et al.,1997;Aggarwal et al.,1990),although a high similarity was exhibited(data not shown).Moreover the nucleotide sequences also showed a high similarityto nucleotide sequence of the dynein heavy chain(XM_001707201,G.lamblia ATCC 50803 dynein heavy chain,Morrison et al.,2007),which is a component protein of dyneins,microtubule mo-tors,and there have been no published reports that dynein heavychain gene is a heat shock induced gene.Only one nucleotide se-quence was found to be different in the forward priming site whencomparing hsp and dynein heavy chain genes.Also reverse primingsequences were comparable between the two sequences(data notshown)implying that similar target sequences affect negatively on378G.-C.Lee et al./Experimental Parasitology 123(2009)377380thebindingspecificityofprimerscausingunreliablePCRamplification.On the basis of phylogenetic analysis of the hsp gene(GenBankaccession No.X16738)using homologues including hsp70 genesand dynein heavy chain genes from various species,the sequenceof the fragment amplified by the Abbaszadegan PCR was clusteredwithdyneinheavychaingene(GenBankaccessionNo.XM_001707201).However,this made a separate branch from theheat shock protein genes of various species including the Giardiahsp70 gene(GenBank accession No.XM_001707918)as presentedin Fig.1.To develop a new RT-PCR to detect G.duodenalis cysts and tosimultaneously define their viability,the hsp70 gene was selectedas a suitable target for the RT-PCR.The hsp70 gene has been usedfor successful detection of viable oocysts of C.parvum and the tran-scription is mainly induced by stress factors including heat shock(Gobet and Toze,2001;Lindquist and Peterson,1990;Stinearet al.,1996).In addition,the hsp70 gene was recently identifiedin G.duodenalis by Morrison et al.(2007)(GenBank accession No.XM_001707918).We designed new PCR primers for the hsp70 gene(GenBankaccession No.XM_001707918)and evaluated whether the newlydeveloped RT-PCR detects the mRNA of the hsp70 indicating geneexpression in viable cysts.The cysts of viable and heat-killed G.duodenalis were serially(10-fold)diluted at the concentration of100104cysts/100ll PBS.To validate that the cysts were dilutedaccurately,the number of cysts were counted at each dilution.Asa result,the cysts were 117.7 7.5/100ll,10.7 0.6/100ll,and0.7 0.6/100ll at 102,101,and 100dilutions,respectively.In orderto inactivate the cysts of G.duodenalis,they were heat-killed at150?C for 15 min as mentioned by Thiriat et al.(1998).Viabilityof the cysts was confirmed by staining and observation proceduresdescribed above in Section 2.The cytoplasm and nucleus werestained by the monoclonal antibody of G.duodenalis conjugatedwith fluorescein isothiocyanate(MAb-FITC)as a green colour andby DAPI as a blue colour in both viable and inactivated cysts,theycould be stained by PI only in the case of inactivated cysts(data notshown).These findings confirm that the viability of G.duodenaliswas completely removed.The amplification in RT-PCR results for the hsp70 was not ob-served with the genetic materials derived from the heat-killed G.duodenalis cysts despite applying heat shock at 45?C for 20 min,indicating that the transcription of hsp70 gene did not occur(Fig.2).In the case of viable cysts of G.duodenalis,it was found thatthe hsp70 mRNA could be detected at 103cysts/100ll without anyheat stimulation(Fig.2).Heat treatment to viable cysts increasedthe detection sensitivity of RT-PCR to as low as 100cyst/100ll,which was attributed to the induction of mRNA transcription ofthe hsp70 gene(Fig.2).These results suggest that one viable cystof G.duodenalis/100ll is detected by using the newly designedRT-PCR.Further,detection sensitivity improved by about 103timesfollowing heat shock treatment.In conclusion,the RT-PCR forhsp70 gene developed in this study presents the potential o