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plants,itisnecessarytodevelopmultiplexPCRfor thesimultaneousdetectionofallfourbananaviruses. Inthisstudy,asimplemultiplexreversetranscrip- tion-PCR(RT-PCR)techniquewasdevelopedand evaluatedforthesimultaneousdetectingallthefour characterizedvirusesofbanana,two(BBrMVand CMV)withssRNAgenomes,one(BBTV)with ssDNAgenomeandone(BSV)withdsDNAgenome. MaterialsandMethods Plantmaterialsandvirusisolates SingleinfectionsofBBTV,BSVandCMVandmixed infectionsofBBTVandBSV,BBTVandCMV,and BSVandCMVinbananaleafsampleswereobtained locally.Inaddition,leafsamplesfromseverallocal bananafarmsandtissue-culturebananaindustries wereusedtovalidatethemultiplexRT-PCR.Single infectionsofBBrMVinleafsampleswerepurchased fromNEOGENorAGDIA(seeTable1).Samples werestoredat ) 70 ° Corasfreezedriedmaterialat ) 20 ° C.Thesesampleswerechosentoreectthe knowngenomicdiversityoftherespectiveviruses (Karanetal.1994;Thomasetal.1997;Paresetal. 1998;Rodonietal.1999). TotalRNAwasextractedfromleaftissues(50mg) ofhealthyandinfectedbananaplantsusinganRNA- isoPlusReagentkit(TaKaRaBiotechnologyCo., Ltd.,Dalian,China)accordingtothemanufacturer Õ s instructions.Thepelletwasresuspendedin50 l lof DEPC-treatedwaterandstoredat ) 20 ° Cuntilused. Designofvirus-specicprimers Atleastthreesetsofprimersweredesignedandevalu- atedforeachvirusbasedonavailablesequencesof BBTV,BSV,CMVandBBrMVinGenBankusingthe PrimerSelectprogramof dnastar dnastar 5.01.(DNASTAR, Inc.,Madison,WI,USA)Afterbeingdesigned,the primersweresynthesizedbyInvitrogenBiotech.Co. Ltd.(Shanghai,China).ForBBTVprimerpairswere designedtoannealtoconservedregionsin RDRP gene accordingtothealignmentofmultiplestrains ⁄ isolates. ForBSVandCMV,primerpairsweredesignedto annealtothe CP gene.ForBBrMV,primerpairswere designedtoannealto HC-Pro gene(Table2).These primerpairsweredesignedtogeneratePCRproducts ofdierentsizesforeachvirusthatwouldbediscern- iblebyagarosegelelectrophoresis.Additionally,Pri- mer premier premier 5.0(Prenierbiosoftinternational,Palo Alto,CA,USA)wasusedtoensurethattheseprimers hadsimilarannealingmeltingtemperatures(Tm)did notformsecondarystructuresanddidnotformpri- mer-dimersduringPCRassays.Potentialinteractions amongprimerswerealsoanalysedusing clustalw clustalw (http://www.ebi.ac.uk/clustalW/index. htm).Informationofpartialprimerpairswasdetailed inTable2. Specicityandcompatibilityofprimerpairs UniplexRT-PCRwasconductedusingeachprimer pairtoevaluateitsspecicityandtodeterminethe mostcommoncyclingconditions.Forrst-strand Table1 QuadruplexRT-PCRanalysis ofeldsamples RegionSamplesBBTVCMVBBrMVBSVBBTV CMVBBTV BSVCMV BSV Qionghai155200100 Chengmai207503001 Wenchang325001000 Haikou45(60) a 10(27)8(6)0(0)6(0)3(0)2(0)1(0) Sanya12(60)0(3)5(5)0(0)0(0)0(0)0(0)0(0) Dongfang2600010000 Ledong181703000 Changjiang176907512 Danzhou(60)(17)(1)(0)(0)(0)(0)(0) Total3658148030934 Percent22.1913.1508.222.470.821.10 a Numbersinparentheseswerecompiledfromtissue-culturesamples. Table2 PartialprimerpairsusedinthemultiplexRT-PCRmethod PrimerSequence(5 ¢ –3 ¢ ) Number ofbaseNucleotideposition a Product length(bp) BBTV-FATGTGGTATGCTGGATGTTC20DNA-1:139–158747 BBTV-RGTTCATATTTCCCGCTTTGA20DNA-1:885–866 CMV-FTATGATAAGAAGCTTGTTTCGCG23RNA3:1551–1573488 CMV-RGCCGTAAGCTGGATGGACAA20RNA3:2038–2019 BBrMV-FCGATACAGAGGGAACCTCTCACCA241921–1944132 BBrMV-RGTCTTGCAGATGGGCTTCGATACTGTG272052–2026 BSV-FGAGACCAAGGTACACAAAATATCATC263138–31631055 BSV-RAACTCTGGTTTTCTTAACTTCTTC244192–4168 ThenumbersindicatethelocationsofdierentprimersonthesequencesoftheircorrespondingviruseswithaccessionNo.NC_003479.1, a NC_001440.1,NC_009745.1andNC_008018.1,respectively. 623 SimultaneousDetectionofAllFourBananaViruses
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