發(fā)展一種新的LAMP技術快速檢測石斑魚虹彩病毒（SGIV）.pdf

1、中山大學碩士學位論文發(fā)展一種新的LAMP技術快速檢測石斑魚虹彩病毒（SGIV）姓名：毛新亮申請學位級別：碩士專業(yè)：海洋生物學指導教師：秦啟偉20070606Developmentofanewmethodofloopmediatedisothermalamplification(LAMPlforra【piddetectionofSingaporegrouperiridovirusfSGIV)廁口r；川小心量bl口，，枷AB：b舳st腳ra

2、c朋t加文矽衙，戶勵fQ；no；we；，均口r；川擁噸量b1芍，，砌略：舳嘲朋加文P衙’尸勵Anewmethodofloopmediatedisothermilamplification皿AMP)wasdevelopedforrapidandsensitivedetectionofSingaporegrouperiridovlrus(SGIV)Asetofsixspecificprimerscontaining2loopprimersw

3、agdesignedbytargetingtheSGIVgenomeDNA(ORF：014L)，arecombinedplasmidpT014LwhichcouldbeuseduspositivecontrolwasconstructedWithBstDNApolymeraselargefragmentwithin20min，thetargetplasmidcanbeamplifiedat65“CTheLAMPproductswerei

4、dentifiedanddigestedwithMst1SGIVwasabletobedetectedin20minsbyusingasetof6primersThedetectiontimewas40rainsusingasetof4primers，Thedetectionlimitswereabout6～600copiesofSGIVgenomicDNAinthreedifferentjudgingmethods：gelelectr

5、ophomsis，SYBRGreenlstainandwhiteprecipitateofmagnesinmpymphosphateComparedtonestedPCRthedetectionlimitofLAMPis1000foldhigherThemwssnoC105Sreactionsin7otherDNAandRNAvirusesindicativeofgoodspecificityoftheLAMETheLAMPmethod

6、wasappliedtodetectSGIVinvimsinfectedGPcellsandgroupertissuesSGIVcouldbedetectedasearlyas45rainininfectedGPcells，TheLAMPdetectionresultsingroupertissuesindicatedthatspleen，liverkidneyandintestineweminfectedbySGW，andspleen

7、andkidneyweremostseriouslyinfectedLAMPwasalsousedt011101“1itorthetranscfiptionandexpressionofORF：014L，Thetestcanbeeasilyusedunderfieldconditionsbecausetheamplificationisabletocarryoutatacertaintemperatureandtheonlyequipm