發(fā)展一種新的LAMP技術(shù)快速檢測(cè)石斑魚(yú)虹彩病毒（SGIV）.pdf

1、中山大學(xué)碩士學(xué)位論文發(fā)展一種新的LAMP技術(shù)快速檢測(cè)石斑魚(yú)虹彩病毒（SGIV）姓名：毛新亮申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：海洋生物學(xué)指導(dǎo)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(lì)fQ；no；we；，均口r；川擁噸量b1芍，，砌略：舳嘲朋加文P衙’尸勵(lì)A(yù)newmethodofloopmediatedisothermilamplification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