SensitiveandFastDNALAMPinanEasy-to-UseMasterMixFormat
Loop-mediatedisothermalamplification(LAMP):Facilitatesrunningamplificationreactionsindifficulttestenvironments,enablinguseoutsideofahigh-techlaboratory.
MasterMixFormat:Simplifiesreactionsetupandminimizesoptimizationrequiredtoproducethebestassayresults.
HighThermalStABIlity:HighlyThermostableenzyme(upto90°Cfor5min)enablespreheatingofreactionscontainingpurifiedtargetDNAwhich,dependingonthetarget,mayincreaseassaysensitivityanddecreasetimetoresults.
ElevatedLAMPReactionTemperature(68-74°C):ImprovesprimerspecificityandreducesbackgroundamplificationdependingontheDNAtarget.
Freeze-dryCompatIBLe:Enablesgenerationofroomtemperaturestabletestkitsthroughlyophilization.
Customizable:OnceprimersareoptimizedwiththeLavaLAMP™DNAMasterMix,wecanworkwithyoutogeneratecustommastermixesfurtheroptimizedforyourtargetand/ortestneeds–contactus
TheLavaLAMP™DNAMasterMixisintendedtosimplifydevelopmentandoptimizationofDNALAMP(loop-mediatedisothermalamplification)reactions.LAMPkitsarecommonlyavailableasmulti-componentkitsthatrequireoptimization(e.g.MgSO4,betaine,enzymeaswellastemperature,primerconcentration,etc.).TheLavaLAMP™DNAMasterMixgreatlysimplifiesreactionoptimizationbylimitingoptimizationtotargetspecificcomponents/conditionssuchasLAMPprimerdesign,targetconcentrationandreactiontemperature.Additionally,thismastermixisheatstableat90°Cfor≤5minutes,whichenablestheadditionofareactionpreheatingstepwhichmay,dependingonthetarget,increaseassaysensitivityanddecreasetimetoresults.Needmoreinformation?SeeourFAQs.
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Figure1.Simplifiedoverviewoftheloop-mediatedisothermalamplification(LAMP)process.Forsimplicity,4/6potentialLAMPprimersareillustrated.Theinclusionoftwoadditionalprimers,F-Loop;ForwardLoopPrimerandB-Loop;BackwardLoopPrimer,oftensignificantlyenhancesamplification.Formoreinformation,pleasevisittheEikenwebsite. |
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Figure2.IllustrationofthedifferencesinLAMPreactionsetupandpotentialoptimizationparameterswhenusingamastermixvs.acomponentkitformat.PanelAillustratesthecomponentsaddedtoaLAMPreaction(left)whenusingtheLavaLAMPDNAMasterMixandthevariousparametersthancanbeoptimized(right).PanelBalsoilllustratesreactionsetup(left)andpotentialoptimizationparameters(right)whenusingastandardLAMPcomponentkit. |
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Figure3.Loop-mediatedisothermalamplification(LAMP)withreal-timefluorescentdetectionofamplifiedproducts.LAMPreactionsweresetupusingtheindicatedkitsaccordingtomanufacturer’srecommendations.TargetDNA(C.difficile)atvaryinginputamounts,tcdAtargetLAMPprimers,andGreenFluorescentDye(LavaLAMPKit)wereincludedinallreactions.ReactionswererunonaCFX96ThermalCycler(Bio-Rad)atthefollowingtemperatures:LavaLAMP;68°C;otherkitsattherecommended65°Candfluorescencewasmeasuredover60minutestodeterminetheTTR.NTC=NoTargetControl. |
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Figure4.TestingthethermostabilityoftheLavaLAMP™DNAMasterMix.TwosetsoftriplicateLAMPreactionsweresetupusingtheindicatedamountsoftargetDNA(C.difficile),tcdAtargetLAMPprimers,andGreenFluorescentDye.Onesetofreactionswaspreheatedto90°Cfor5minutes,andthenallreactionswereincubatedfor60minutesonaCFX96ThermalCycler(Bio-Rad)at68°CandfluorescencewasmeasuredduringthecourseofthereactionstodeterminetheTTR.NTC=NoTargetControl. |
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Figure5.TestingtheperformanceoflyophilizedLavaLAMP™DNAMasterMix.TheLavaLAMPDNAMasterMix(12.5µL)wasaliquotedintotubesandlyophilizedusingaVirTisWizard2.0.Afteroneday,eachlyophilizedtubewasreconstitutedin12.5µLwater.ThensixreplicateLAMPreactionsperconditionweresetupusingthereconstitutedlyophilizedmastermixandthestandardLavaLAMPDNAMasterMix(Controls).TheindicatedamountsoftargetDNA(S.aureus),clfAtargetLAMPprimers,andGreenFluorescentDyewereaddedtothereactions.Allreactionswereincubatedfor60minutesinaCFX96ThermalCycler(Bio-Rad)at68°CandfluorescencewasmeasuredduringthereactionstodeterminetheTTR.NTC=NoTargetControl.
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Figure6.Analysisofdaytodayvariability.Onthreedifferentdays,sixreplicateLAMPreactionsweresetupperconditionusingtheindicatedamountsoftargetDNA(M13mp18),M13mp18targetLAMPprimers,andGreenFluorescentDye.OneachdaythereactionswererunonaCFX96ThermalCycler(Bio-Rad),amplificationwasmonitoredinreal-time,andthenresultswereanalyzedtodeterminetheTTR.NTC=NoTargetControl. |
ORDERINFORMATION
BothLavaLAMP™DNAMasterMixkitscontain:LavaLAMPDNAMasterMix,DNAPositiveControlLAMPPrimerMix,andDNAPositiveControl.TheLavaLAMPDNAMasterMixwithDyealsocontainsGreenFluorescentDyeforfluorescentdetectionofamplifiedDNA.
Licensinginformation:LucigenisafullylicensedproviderofLAMPreagentsforresearchuse.PatentsWO00/28082,WO01/34790,andWO01/77317regardingtheLAMPmethodareownedbytheEikenChemicalCo.Ltd.LavaLAMP™,OmniAmp®andBstPolymerase,ExonucleaseminusaresoldbyLucigenunderlicenseforuseinLAMPforresearchuseonly.TheproductsmaynotbeusedforLAMP-basedhumanordiagnosticpurposeswithoutobtainingalicensefromEiken.USPatent8093030forLavaLAMPandOmniAmpisownedbyLucigenCorp.
Itisthesoleresponsibilityofthebuyertoensurethatuseoftheproductdoesnotinfringethepatentrightsofthirdparties.Ifthepurchaserisnotwillingtoaccepttheseuselimitations,LucigenCorporationiswillingtoacceptreturnoftheproductforafullrefund.
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我做的是用锁式探针来检测dna模板的ram，探针80base，其中杂交区40base，引物分别为16、18base，连接酶选取的T4 ligase和Taq DNA ligase两种，分别对应恒温和变温情况下的扩增。水解体系为EXO I和Exo Ⅲ的混合体系，参考的外文文献，应该没什么问题。聚合酶用的是Bst DNA大片段聚合酶。
一开始选择的探针浓度为200pmol/L，扩增效果挺好，当时用的DNA模板是质粒阳性标准品，但是后来在做敏感性的时候发现条带梯度差异几乎没有，而且阴性也出带，后来就没法做敏感性和特异性验证了，实验停滞了2个月一直也没进展。
按理说RAM借助锁式探针扩增的独特方式以及水解体系的存在可以有效防止污染才对，希望大家给点建议该如何解决。
万分感谢！！！！！！！！！！
附一张以前做敏感性的图（marker为2000的DNA ladder）以做参考
(2)操作多份样品时，应先配制反应混合液并分装，减少操作，避免污染，同时增加反应的精确度。试剂的配置和分装应在装有紫外灯的超净工作台或负压工作台操作。
(3)移液器和吸头需专用。由于移液器极易受气溶胶或模板核酸的污染，移液器应尽可能使用可替换或可高压处理的，吸头尽可能使用带滤芯的吸头。
(4)防止操作人员污染，使用一次性手套，EP管与吸头应一次性使用，吸头不要长时间暴露于空气中，避免气溶胶的污染。
(5)加样或吸取模板核酸时要十分小心，吸样要缓慢，防止样品进入移液器内；吸样时尽量一次性完成，忌多次抽吸，以免交叉污染或产生气溶胶污染。
(6)EP管打开应先离心，将管壁及管盖上的液体甩至管底部。开管动作要轻，以防管内液体溅出。若不小心溅到手套或桌面上，应立刻更换手套并用稀酸擦拭桌面。
(7)模板核酸应密封保存，防止外溢及外来核酸的进入。
(8)设立适当的阴阳性对照及重复实验，既可验证LAMP反应的准确性，又可以协助判断扩增系统的可信性。
(9)扩增产物应妥善处理，尽量避免开盖检测，开盖极易产生气溶胶污染。本公司的试剂均采用闭管检测，有效防止污染发生。展开
同一温度下，首先通过M-MLV反转录酶产生靶标核酸（RNA）的一个双链DNA拷贝，然后利用T7RNA多聚酶从该DNA拷贝上产生多个（100～1000个）RNA拷贝；每一个RNA拷贝再从反转录开始进入下一个扩增循环；还可以加入荧光探针（分子信标），带有荧光标记的探针和这些RNA拷贝特异结合，产生荧光。该荧光信号可由实时荧光PCR仪实时捕获，直观反映扩增循环情况。
SAT检测技术的优势
→先进的恒温扩增技术
核酸扩增在一个温度下进行（42℃），无需热循环。
→领先的RNA检测技术
SAT技术直接以病原体特异性RNA为扩增靶标，以扩增产物RNA为检测靶标，在实际应用中更显优势意义。
→更高的检测灵敏度和准确性
SAT技术的扩增效率极高，15～30分钟即可将模板扩增109倍，检测灵敏度和准确性远高于其他核酸检测技术。扩增时间短，效率高。
→有效减少假阴性结果
SAT技术采用M-MLV逆转录酶和T7RNA多聚酶进行核酸扩增，相对于其它核酸扩增技术，反应抑制物更少，有效减少假阴性结果。
→有效的解决了扩增产物的污染问题
由于扩增产物为RNA，环境中极易降解，从而大幅度提高检测结果的可靠性。
→大大降低了核酸扩增实验室的要求
