Detection of foot-and-mouth disease virus rna by reverse transcription loop-mediated isothermal amplification

Chen Hao-Tai, Jie Zhang, Liu Yong-Sheng, Liu, Liu Xiang-Tao

Description

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for foot-and-mouth disease virus (FMDV) RNA. The amplification was able to finish in 45 min under isothermal condition at 64°C by employing a set of four primers targeting FMDV 2B. The assay showed higher sensitivity than RT-PCR. No cross reactivity was observed from other RNA viruses including classical swine fever virus, swine vesicular disease, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus. Furthermore, the assay correctly detected 84 FMDV positive samples but not 65 FMDV negative specimens. The result indicated the potential usefulness of the technique as a simple and rapid procedure for the detection of FMDV infection.

Sujets

• Foot-and-mouth disease virus
• Detection
• Sensitivity
• Specificity

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 29
• Langue: English

Extrait

Chen
etal
.
VirologyJournal
2011,
8
:510
http://www.virologyj.com/content/8/1/510

RESEARCH

OpenAccess

Detectionoffoot-and-mouthdiseasevirusrnaby
reversetranscriptionloop-mediatedisothermal
amplification
Hao-taiChen
†
,JieZhang
†
,Yong-shengLiu
*
andXiang-taoLiu
*

Abstract
Areversetranscriptionloop-mediatedisothermalamplification(RT-LAMP)assaywasdevelopedforfoot-and-mouth
diseasevirus(FMDV)RNA.Theamplificationwasabletofinishin45minunderisothermalconditionat64°Cby
employingasetoffourprimerstargetingFMDV2B.TheassayshowedhighersensitivitythanRT-PCR.Nocross
reactivitywasobservedfromotherRNAvirusesincludingclassicalswinefevervirus,swinevesiculardisease,porcine
reproductiveandrespiratorysyndromevirus,Japaneseencephalitisvirus.Furthermore,theassaycorrectlydetected
84FMDVpositivesamplesbutnot65FMDVnegativespecimens.Theresultindicatedthepotentialusefulnessof
thetechniqueasasimpleandrapidprocedureforthedetectionofFMDVinfection.
Keywords:
Foot-and-mouthdiseasevirus,Detection,Reversetranscriptionloop-mediatedisothermalamplification,
Sensitivity,Specificity

1.Background
assaysweretime-consumingandlaborious,which
Foot-and-mouthdiseasevirus(FMDV)isamemberofrequiredcentralizedlaboratoryfacilitiesandclinicalspe-
thegenus
Aphthovirus
ofthefamily
Picornaviridae
,cimensubmissions,resultedinthedelayofFMDVdiag-
whichisdividedintosevenserotypeswithnocross-pro-nosis.Giventheseproblems,arapid,simpleand
tectionconferredamongtheserotypes[1].DuetothepracticalassaytodetectFMDVinanimalanditspro-
aggressivenatureoffoot-and-mouthdisease(FMD),ductswasthereforerequiredinclinicalpractice.
outbreaksusuallyresultinsevereeconomiclossesandAreversetranscriptionloop-mediatedisothermal
impactonbothnationalandinternationaltradewithinamplification(RT-LAMP)wasappliedsuccessfullyto
thelivestockandanimalproducts[2].Rapidandaccu-thedetectionofmanyanimalviruses[6-9].Inthis
ratediagnosisofanysuspectedFMDcasesisofutmoststudy,weevaluatedthepotentialofRT-LAMPforthe
urgencytocontrolthisveterinaryinfectiongiventhedevelopmentofasimpleandrapiddetectionsystemfor
extremecontagiousnessofthecausativevirus.FMDVRNA.
ConventionallaboratorydiagnosisofFMDwasmade
byELISAdetectionofspecificviralantigensandby
2.Materialsandmethods
observationofcytopathiceffectsincellculture[3].
2.1.Viralstrainsandsamples
Alternatively,theconventionalreversetranscriptaseThestrainsO/CHA/1999,A/CHA/2009,C/SU/1958,
polymerasechainreaction(RT-PCR)[4]andreal-timeAsia1/CHA/2005weredevelopedRT-LAMPmethod
RT-PCR[2,5]weredevelopedtocomplementprimaryandthenthestainO/CHA/1999wastotestthedetec-
diagnostictechniquesfortheFMDVinfection.Thesetionlimit.TheserotypesOandAsia1werepropagated
inIBRS-2cells,andtheserotypesAandCwereinocu-
*Correspondence:[e-mail];[e-mail]
latedin3-daysucklingmice,respectively.
†
Contributedequally
Otherfieldisolatesincludingclassicalswinefever
StateKeyLaboratoryofVeterinaryEtiologicBiology,NationalFoot-and-
virus(CSFV),swinevesiculardiseasevirus(SVDV),por-
MouthDiseaseReferenceLaboratoryofChina,KeylaboratoryofAnimal
VirologyofMinistryofAgriculture,LanzhouVeterinaryResearchInstitute,
cinereproductiveandrespiratorysyndromevirus
ChineseAcademyofAgriculturalSciences,Lanzhou,730046,Gansu,P.R.
China

©2011Chenetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.

Chen
etal
.
VirologyJournal
2011,
8
:510
http://www.virologyj.com/content/8/1/510

Table1ResultcomparisonofRT-LAMPandRT-PCRassaysusing139samples
PathogenStrain(specimennumber)Results(positivenumber/specimennumbertested)
RT-LAMPRT-PCR
FMDVO/CHA/1999(N=32)+(32/32)+(32/32)
A/CHA/2009(N=22)+(22/22)+(22/22)
Asia1/CHA/2005(N=20)+(20/20)+(20/20)
C/UN/1988(N=10)+(10/10)+(10/10)
CSFVC2008(N=17)
–
(17/17)
–
(17/17)
SVDVSVDV01(N=10)
–
(10/10)
–
(10/10)
PRRSVHPBEDV(N=19)
–
(19/19)
–
(19/19)
JEVJEV2009(N=9)
–
(9/9)
–
(9/9)
+,positivereaction;
–
,negativereaction

Page2of4

(PRRSV)andJapaneseencephalitisvirus(JEV)were(SAT2),AY593850(SAT3),AY593852(SAT3),
identifiedbyRT-PCR.AY593853(SAT3).
Atotalof139samplesincluded84FMDV,17CSFV,RT-LAMPreactionwascarriedoutinaconventional
10SVDV,19PRRSVand9JEVspecimens,whichwerewaterbathbymixing2.0
μ
MeachofFIPandBIPpri-
alsoidentifiedbyRT-PCR(Table1).mer,0.2
μ
MeachofFandBprimer,1.0mMeach
deoxynucleosidetriphosphate,8UofBstDNApolymer-
2.2.RNAextraction
ase(NewEnglandBiolabs)and1UoftheTHERMO-X
RNAwasextractedfromFMDV-infectedandhealthyreversetranscriptase(Invitrogen)usingthemanufac-
animals,usingaRNeasyMiniKit(Qiagen)accordingtoturer
’
ssupplied10×buffer(containing4mMofMgSO
4
,
themanufacturer
’
sinstructions.Afterextraction,RNA0.8Mbetaine)and1
μ
lofextractedtemplateRNAina
waselutedin60
μ
lofelutionbufferandstoredat-20°C.0.2mlEppendorftube.Theamplificationwasperformed
at64°Cfor45minandthenterminatedbyheatingat
2.3.ConventionalRT-PCRandRT-LAMP
80°Cfor10min.RT-LAMPproductswereanalyzedby
ThedetectionofFMDVbyRT-PCRwasperformedwith2.5%agarosegelelectrophoresis.
primersdescribedpreviously[10].Asetoffourprimers,
F,B,FIPandBIPweredesignedbytargetingconserved
2.4.SensitivityandspecificityofRT-LAMPforFMDV
regionsofFMDV2B(Table2).2BnucleotidesequencesComparedtoRT-PCR,thedetectionlimitofRT-LAMP
ofalltheFMDVserotypeswereretrievedfromGenBankwastestedusingthesametemplatesatidenticalconcentra-
andalignedusingthesoftwareprogramDNAStartions,whichwasperformedintriplicateateachconcentra-
(DNASTAR,Inc.Madison).Theaccessionnumberstionoftemplates.TheO/CHA/1999RNAwasquantitated
usedforthealignmentwerethefollowing:AY593782usingUVspectrophotometry(UNICAM3000,US).Serial
(A),AY593801(A),AY593768(A),AY593795(Asia1),dilutionsof1,10,10
2
,10
3
and10
4
copiesperreactionfrom
EF149010(Asia1),AY593796(Asia1),AY593799(AsiaFMDVstrainswereusedintheassay.Toassessthespecifi-
1),AY593810(C),AY593806(C),AY593809(C),cityofRT-LAMP,crossreactionswithRNAofthestrains
AJ539138(O),AY593817(O),AF511039(O),AY593845CSFV,SVDV,PRRSVandJEVwereexamined.ViralRNA
(SAT1),AY593839(SAT1),AY593844(SAT1),ofthestrainsO/CHA/1999,A/CHA/2009,C/SU/1958and
AY593847(SAT2),AY593848(SAT2),AY593849Asia1/CHA/2005wasusedasthepositivecontroland
RNAextractedfromhealthyswinetissueswasusedasthe
negativecontrol.Inaddition,RNAfrom139sampleswas
Table2DetailsofRT-LAMPprimersdesignedfor
extractedandsubjectedtoFMDVRT-LAMP.
detectionof2BcodingsequencesofFMDV
PrimernameSequence
3.Results
F5
’
-CCTGTCGTGCATGGCCGCTGT-3
’
3.1.AnalysissensitivityoftheLAMPmethodcomparedto
PCR.DetectionlimitofFMDVRT-LAMP
B5
’
-GAAACACGAGGCAACTTTGAC-3
’
TheamplificationbyFMDVRT-LAMPshowedalad-
FIP5
’
-CTTACAGACGAAGGTGCTGTC
der-likepattern(Figure1).Theresultindicatedthatthe
+CATCATGCTGGCCGACACCG-3
’
detectionlimitofFMDVRT-LAMPwas10copies
BIP5
’
-AGATCTCCGACTCGCTCTCCA
whereasthatofRT-PCRwas100copiesperreaction.
+
ThedetectionsensitivityofFMDVRT-LAMPwas
ACAGGACCGGTGCTCCGAAAC-3
’
thereforegreaterthanthatofRT-PCR.

Chen
etal
.
VirologyJournal
2011,
8
:510
http://www.virologyj.com/content/8/1/510

Figure1
AgarosegelelectrophoresisanalysisofRT-LAMP
productsusingFMDVreferencestrains
.LaneM,DNAMarker
DL2000(2000,1000,750,500,250,100bp);Lane1,PRRSV;Lane2,
O/CHA/1999;Lane3,A/CHA/2009;Lane4,Asia1/CHA/2005.
3.2.AnalyticalcrossreactionofFMDVRT-LAMP
RNAextractedfromtissuesofhealthyanimalsandpigs
infectedwithCSFV,SVDV,PRRSVandJEVwasusedas
templatesforRT-LAMP.Agarosegelelectrophoresis
analysisindicatedthatFMDVRT-LAMPreactiondid
notdetectCSFV,SVDV,PRRSVandJEVaswellasgave
anegativereactionwithtissuesofhealthyswine,but
FMDVRNAwasthepositivereaction.
3.3.EvaluationofFMDVRT-LAMPwithsamples
ToevaluatespecificityandsensitivityofFMDVRT-
LAMP,theassaycorrectlydetected84FMDVpositive
samplesbutnot65FMDVnegativespecimens(Table1).
4.Discussion
ForcountriestoremainFMD-freeortocontrolFMD,
rapidandaccuratedetectionofFMDVwasplayedacri-
ticalroleintheimplementationofeffectivecountermea-
surestocontrolspreadofFMD.RT-LAMPwasa
sensitivediagnosticmethod,whichwasquitesimple,
requiringonlyaconventionalwaterbathorheatblock
forincubationunderisothermalconditions[7-9].
AnotherusefulfeatureofRT-LAMPisthatitsproducts
canbeobserveddirectlybynakedeye,becauseawhite
precipitateofmagnesiumpyrophosphateformsinthe
reactiontube[11].AddingSYBRGreenItoRT-LAMP
reactionscanalsoincreasetheeasedetectionbythe
nakedeye[12].
UnlikeRT-PCR,thegreatersensitivityandspecificityof
RT-LAMPwerereportedtoutilizetodetectanimal
viruses[7,8].Thelackofcrossreactionobservedwith
CSFV,SVDV,PRRSVandhost-derivedRNAindicated
thatFMDVRT-LAMPassaywasspecificinadditionto
highsensitivity.In