LAMP-LFD-技术--文件课件.pptx

1、LAMP-LFD无电进行LAMP反应PMA消除LAMP产物污染2015实验计划LAMP反应体系的优化试纸条的搭建双重检测体系的建立快速多重LAMP-LFD检测体系的构建LAMP反反应体系应体系的优化的优化Bst酶预变性步骤DNA快速提取煮沸法提取革兰氏阴性菌及革兰氏阳性菌（加入溶菌酶）取消95度5min预变性步骤试纸条的搭建原金1:1稀释Tris量优化：20mM，50mM，100mM，200mM1:1稀释原金PVP量优化：0，0.5%，1.0%，1.5%，2.0%原金1:1稀释BSA量优化：0，0.5%，1.0%，2.0%酪蛋白量优化：0，0.05%，0.1%，0.15%，0.20%Tween

2、-20量优化：0，0.5%，1.0%，1.5%，2.0%海藻糖量优化：0，1.0%，2.0%，3.0%，4.0%试纸条的搭建一号胶体金最佳0.2M 碳酸钾量：3ul最小抗体量：2ul二号胶体金最佳0.2M 碳酸钾量：4ul最小抗体量：2ul试纸条的搭建两种新的胶体金进行抗体标记两种新的胶体金进行抗体标记量和最佳量和最佳PH值的优化值的优化2号金32nm-59nm试纸条的搭建T线线PH值值优化优化试纸条初步搭建完成试纸条初步搭建完成一号胶体金二号胶体金三号胶体金1号：原金标抗体（阴性）；2号：1：1稀释抗体（阴性）；3-7号：1：1稀释抗体（LAMP产物量1ul、0.1ul、0.01ul、0.0

3、01ul、0.0001ul）试纸条的搭建123 456712 345671234567试纸条的搭建胶体金的制作胶体金的制作氯金酸氯金酸工作液浓度为工作液浓度为1%工作液浓度为工作液浓度为1%柠檬酸三钠柠檬酸三钠煮沸，定容获得大煮沸，定容获得大小不同的胶体金小不同的胶体金不同量不同量1%柠柠檬酸三钠檬酸三钠100ml 0.01%氯金酸氯金酸1ml,1%氯金酸氯金酸+4ml,1%柠檬酸三钠（约柠檬酸三钠（约10nm胶体金）胶体金）1ml,1%氯金酸氯金酸+1.5ml,1%柠檬酸三钠柠檬酸三钠（约（约24.5nm胶胶体金）体金）1ml,1%氯金酸氯金酸+0.42ml,1%柠檬酸三钠柠檬酸三钠（约（约

4、97.5nm胶胶体金体金）约约10nm胶体金胶体金约约97.5nm胶体金胶体金约约24.5nm胶体金胶体金双重检测体系的建立抗地高辛抗体质量：国产抗抗地高辛抗体质量：国产抗地高辛抗体更换为地高辛抗体更换为sigma公公司司D8156(anti-Digoxin)1号：商业化条；2号4号：商业化C、T线+自制0号、1号、2号胶体金制作金标垫；检测5ul沙门氏菌LAMP产物1234金标抗体浓度问题：复溶金标抗体浓度问题：复溶液、稀释液重新配置仍无液、稀释液重新配置仍无明显改进。购买新的明显改进。购买新的FITC抗抗体体Multiplex LFDLAMP扩增目的片段的筛选Multiplex LAMP双

5、重检测体系的建立#NAME?基因检测基质检测方法文章第一作者出版年份文章名称L.monocytogenesiapfood juiceKalliopi Rantsiou2012Strain dependent expression of stress response and virulence genes ofListeriamonocytogenesin meat juices as determined by microarrayL.monocytogenesssrA soft cheese,meat,milkRT-PCRJustin O Grady2008Rapid real-time

6、PCR detection ofListeria monocytogenesin enrichedfood samples based on the ssrAgene,a novel diagnostic targetL.monocytogenes ATCC19115 hlyA foodRT-LAMPXiaoxiao Shan2012Rapid Detection of Food-borne Listeria monocytogenesby Real-time Quantitative Loop-mediated Isothermal AmplificationL.monocytogeness

7、srA Fraser brothRT-PCRJustin OGrady2009Rapid detection ofListeria monocytogenesin food using cultureenrichment combined with real-time PCRL.monocytogenes hlyA raw meat and meat products2014Prevalence and antimicrobial susceptibility of Listeriamonocytogenes andmethicillin-resistantStaphylococcusaure

8、usstrains from raw meat and meat products in Zaria,NigeriaL.monocytogenes hlyAinfant formula and lettuce 2014Real-time PCR detection ofListeria monocytogenesininfant formula and lettuce following macrophage-basedisolation and enrichmentL.monocytogenes hlyAaccession number M24199Grazia Marina Quero20

9、14Quantitative detection ofListeria monocytogenesin raw milk and softcheeses:Culture-independent versus liquid-and solid-based culturedependent real time PCR approacL.monocytogenesHly-f(ACT TCG GCG CAA TCA GTG A)andHly-r(TTGCAA CTG CTC TTT AGT AAC AGC TT)chilled porkRT-PCRKeping Ye2012Rapid detectio

10、n of viableListeria monocytogenesin chilled pork by real-timereverse-transcriptase PCRL.monocytogene hlyA raw shrimp a multiplex real-time PCRZhaohuan Zhang2015Development of a multiplex real-time PCR method for simultaneousdetection ofVibrio parahaemolyticus,Listeria monocytogenesandSalmonellaspp.i

11、n raw shrimpL.monocytogenehlysoft cheese real-time PCRMonica Virginia Gianfranceschi2014European validation of a real-time PCR-based method for detection ofListeria monocytogenesin soft cheeseL.monocytogeneshlyALAMPLi Wang2012Development and application of a simple loop-mediatedisothermal amplificat

12、ion method on rapid detection of ListeriamonocytogenesstrainsL.monocytogenesprfA fooda combinedenrichment/real-time PCR methodPeter Rossmanith2006Detection ofListeria monocytogenesin food using a combinedenrichment/real-time PCR method targeting the prfA geneL.monocytogeneshylA raw and ready-to-eat

13、foodsMPN-PCR detectionM.N.Marian2012MPN-PCR detection and antimicrobial resistance ofListeria monocytogenesisolated from raw and ready-to-eat foods in MalaysiaL.monocytogenesiap deli meatsa new multiplex PCR Haiquan Liu2015Rapid detection and differentiation ofListeria monocytogenesandListeriaspecie

14、s in deli meats by a new multiplex PCR method单增李斯特菌:HlyA菌种检测方法目的基因检测基质出版年份 注释文章第一作者 文章名称Staphylococcus aureus mLAMPnuc 耐 热 核 酸 酶(Genebank NC_002952.2)酶切位点的选择2012mLAMP-PCR-sequencing strategy was applied to analyze the mLAMP productsKan Jiang A novel,sensitive,accurate multiplex loop-mediated isother

15、mal amplification method for detection of Salmonella spp.,Shigella spp.and Staphylococcus aureus in food aptamer2012Nuo DuanDual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcu

16、s aureusMcat2014S.aureus是革兰氏阳性菌Bst酶超过70度会不可逆失活MohammadaliSafaviehHigh-throughput real-time electrochemical monitoring of LAMPfor pathogenic bacteria detectionbulk milk2011Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark:a de

17、scriptive studyPCR16S rRNA(FJ907240.1)2012同时检测四种菌，在16srRNA中找到对齐部分Nguyen Tien HuyDevelopment of a single-tube loop-mediated isothermalamplification assay for detection of four pathogens of bacterial meningitisnuc（DQ507382.1）nuc encodes the thermostable nuclease 2015MRSA和普通金葡的区别金葡总污染奶在奶中，其他非目标菌不会有影响Zh

18、ihong ZhangPropidium monoazide combined with real-time PCR for selective detection of viable Staphylococcus aureus in milk powder and meat productsChen.(2012)mPCR 16S rDNAFusco.(2011)qPCR entero toxin gene cluster(egc)2015Jodi WoanFei LawRapid methods for the detection of foodborne bacterialpathogen

19、s:principles,applications,advantages andlimitationsqPCRSa4222014在猪肉中的检测Kai MaRapid and simultane ous detection of Salmonella,Shigella,and Staphylococ cus aureus in fresh pork using a multiplex real-time PCR assay base d on immunomagne tic separationseg(19.7%),sej(16.2%),see(12.8%),sea(11.1%),seb(10.

20、3%),eta,etband tsst-1 genes were not detected2015detection of genes encodingstaphylococcal enterotoxins,exfoliative toxins A and B(eta and etb),toxic shock syndrome toxin 1(tsst-1),and resistance to methicillin-oxacillin(mecA).Hao,DanPrevalence,Toxin Gene Profiles,and Antimicrobial Resistance of Sta

21、phylococcus aureus Isolated from Quick-Frozen Dumplings mecA(D86934.2)(20-base gene sequence)2014Jingyu ShiA fluorescence resonance energy transfer(FRET)biosensor based on graphene quantum dots(GQDs)and gold nanoparticles(AuNPs)for the detection of mecA gene sequence of Staphylococcus aureusmecA2014

22、Fast detection and differentiation of S.aureus,CoNSDifferentiation Between Staphylococcus Aureus and Coagulase-Negative Staphylococcus Species by Real-Time PCR Including Detection of Methicillin Resistants in Comparison to Conventional Microbiology Testing金黄色葡萄球菌:nuc 沙门氏菌:siiA菌种基因检测基质检测方法文章第一作者出版年份文

23、章名称SalmonellainvA(GenBank accession no.NC_003197)ofSalmonella and ipaH(GenBank accession no.M32063)milkmultiplex loop-mediated isothermal amplification-RFLPYanchun Shao2011Development of multiplex loop-mediated isothermal amplification-RFLP(mLAMP-RFLP)to detectSalmonellaspp.andShigellaspp.in milkSal

24、monellainvAa multiplex real-time PCR method Alejandro Garrido2013In-house validation of a multiplex real-time PCR method forsimultaneous detection ofSalmonellaspp.,Escherichia coliO157andListeria monocytogenesS.TyphimuriumfliCfood productsmultiplex PCR assayYoujun Yang2013Magnetic nano-beads based s

25、eparation combined with propidiummonoazide treatment and multiplex PCR assay for simultaneousdetection of viableSalmonellaTyphimurium,Escherichia coliO157:H7 andListeria monocytogenesin food productsSalmonellainvA foodLAMPYuxin Ye2012Application ofin situ loop-mediated isothermal amplification metho

26、d fordetection ofSalmonellain foodsS.TyphimuriumfliCfoodPCRYoujun Yang2012Development of a multiplexed PCR assay combined with propidium monoazidetreatment for rapid and accurate detection and identification of three viableSalmonella entericaserovarsSalmonellafimYfoodLAMPYaoqi Zhang2012Development o

27、f afimY-based loop-mediated isothermal amplification assay fordetection ofSalmonellain foodSalmonellainvAProduceLAMPSiyi Chen2011Rapid Detection of Viable Salmonellae in Produce by CouplingPropidium Monoazide with Loop-Mediated Isothermal AmplificationSalmonellainvAprocessed food samplesa multiplex

28、real-time PCR method Alejandro Garrido2012Development of a multiplex real-time PCR method for simultaneous detection ofSalmonella enterica,Shigella XexneriandListeria monocytogenesin processed food samplesSalmonellainvAPCRA.Banihashemi2012Long-amplicon propidium monoazide-PCR enumerationassay to det

29、ect viableCampylobacterandSalmonellaSalmonella iroBBloodPCRVithiya GaneSan2014Detection of Salmonellain Blood by PCR using iroBgeneS.entericaCMCC 50041orgCraw shrimp real-time PCRZhaohuan Zhang2015Development of a multiplex real-time PCR method for simultaneousdetection ofVibrio parahaemolyticus,Lis

30、teria monocytogenesandSalmonellaspp.in raw shrimpSalmonellayrfHChicken Meat InsulatedIsothermal PCRHAU-YANG TSEN2013Detection ofSalmonellain Chicken Meat by InsulatedIsothermal PCRSalmonellasifBGenBank:AF236076.1Artificially Contaminated BeefPolymeraseChain ReactionMichelle Vieira de Almeida2014Eval

31、uation of Target Sequences for the PolymeraseChain ReactionBased Detection ofSalmonellain Artificially Contaminated BeefSalmonellasiiAGenbank accession:AJ576316Real time PCRAmal Ben Hassena2015Real time PCR gene profiling and detection ofSalmonellausing a noveltarget:ThesiiAgeneSalmonellsiiAThorsten

32、 Wille2014SiiA and SiiB are novel type I secretion systemsubunits controlling SPI4-mediated adhesion ofSalmonella enterica阪崎克罗诺杆菌:ITS或fusA 菌种检测方法目的基因检测基质出版年份 注释文章第一作者 文章名称Enterobacter sakazakii(Cronobacter sp.)cross-priming amplification(CPA)-LFD 16S-23S rDNA internal transcribed spacerE.sakazakii(G

33、enBankAY702093.1)polluted powdered infant milk formula2010Zhao YulongRapid and sensitive detection of Enterobacter sakazakii by cross-priming amplification combined with immuno-blotting analysisPCR16S rRNA gene-2003、2004the 16S-23S rDNA(ITS)-2006ompA-2006rpoB-2009powdered infant formula(PIF）2014 HUI

34、 LIThe Cronobactersp.in milk and dairy products:Detection and typingPCRompA(DQ000206)powdered milk products2014说明OmpA与治病有关Jennifer ZimmermannDevelopment of a rapid detectio n system for opportunistic pathogenic Cronobacter spp.in powdered milk productsLAMPinternal intergenicspacer(IGS)sequence16S/23

35、S IGSs specific sequence(GI:AY702093.1)powdered milk products2012Xu LiuDevelopment of a loop-mediated isothermal amplification assayfor detection of Cronobacterspp.(Enterobacter sakazakii)LAMP ompA(GenBank accession no.DQ000206)powdered infant formula(PIF)2012Hongying FanDevelopment of a Loop-Mediat

36、ed Isothermal AmplificationAssay for Sensitive and Rapid Detection of Cronobacter sakazakiiPCRgyrB（gyrB Nos.JX983606JX983643 and JX983649JX983653.）powdered infant formula2013说明16srRNA的不足Wanyi ChenDevelopment of a PCR assay for rapid detection of Cronobacter spp.from foodPCR16S and 23S rDNA(ITS)IGSgl

37、uA（Lehneret al.）plants2013Mouhammad BelalDetection of Cronobacter spp.(formerly Enterobacter sakazakii)from medicinal plants and spices in Syria reverse transcription polymerase chain reaction(RT-PCR）mRNAdry aquatic products2012Luria-Bertani(LB)broth at37C for 16 to 18 h to 108CFU/mLYingwang YeDetec

38、tion of Viable Cronobacterspp.(Enterobacter sakazakii)by One-Step RT-PCR in Dry Aquatic Productreal-time PCR-based microfluidics platformreconstitutedmilks2013阪崎污染种类列举很多煮沸法提取DNAWalid M.El-SharoudA real-time PCR-based microfluidics platform for the detection of Cronobac ter sakazakii in reco nstitute

39、d milksprobe-magnetic separation pCr（PMS-PCR）16S rRNAinfant formula samples2014nucleic acid probe combined with magnetic beads Feng XuDetection of Cronobacter species in powdered infant formula by probe-magnetic separation pCr PCR16S rDNA201416S rDNA(1.5 kb)(fmb01efmb13)Yuanhong LiIsolation,ide ntif

40、ication and antimicrobial resistance of Cronobacter spp.isolated from various foods in Chinaduplex PCR combination with(CELIF)16S23S rDNA ITSOmpAinfant food formula2013煮沸法提DNA说明ITS很好Jia RuanRapid and sensitive detection of Cronobacter spp.(previously Enterobacter sakazakii)in food by duplex PCR comb

41、ined with capillary electrophoresislaser-induced fluorescence detectorLAMP引物的设计Multiplex LAMP双重检测体系的建立 单增李斯特菌 ：3组常用引物+3组单环引物 沙门氏菌：4组常用引物金黄色葡萄球菌：3组常用引物+2组双环引物阪崎克罗诺杆菌：2组常用引物制得PCR及LAMP产物测量产物OD值以得到产物浓度应用PMA处理LAMP产物黑暗条件下孵育光照处理将处理后的产物按照模板量进行LAMP扩增不同的方法进行检测确定PMA用灭菌水和DMSO溶解的区别确定光照时是否需要冰上处理确定最佳的PMA工作液浓度确定最佳的

42、曝光时间确定最佳的曝光距离确定最佳的孵育时间确定室内普通光照和操作间的光照区别优化PMA处理PCR条件 确定最佳的PMA工作液浓度无光照时有无光照时有类似提取基类似提取基因组因组DNA大大小的条带小的条带以DNA为模板，加入不同浓度PMA以PCR产物为模板，加入不同浓度PMA无光照无光照优化PMA处理LAMP条件阴性、8ulPMA、6ulPMA、4ulPMA、2ulPMA、0ulPMA、LAMP为模板阳性、Marker文献报道过文献报道过PMA会与水反应，会与水反应，但是此实验结果有不同的但是此实验结果有不同的结果，结果，或是超过了能反应的比例或是超过了能反应的比例阴性、8ulPMA、6ulP

43、MA、4ulPMA、2ulPMA、稀释10倍的PMA8ul、6ul、4ul、2ul、LAMP为模板阳性、DNA模板应用原有的PMA进行预实验，验证PMA对LAMP产物存在作用新定PMA用水稀释成20mM存储浓度阴性、20mM浓度PMA8ul、6ul、4ul、2ul；LAMP为模板阳性、DNA模板阳性优化PMA处理条件抑制效果较原有PMA更加明显优化PMA处理条件阴性、2mMPMA7ul（无冰、有冰）、5ul（无冰、有冰）、3ul（无冰、有冰）、0ul（LAMP模板、DNA模板）阴性、2mMPMA1ul（无冰、有冰）、2ul（无冰、有冰）、4ul（无冰、有冰）、0ul（LAMP模板、DNA模板）

44、出现了较为明显的污染阴性、DNA模板、LAMP产物模板、2mMPMA1ul（无冰、有冰）、2ul（无冰、有冰）、3ul（无冰、有冰）、4ul（无冰、有冰）、5ul（无冰、有冰）、6ul（无冰、有冰）、7ul（无冰、有冰）、MarkerReal-time LAMP 扩增沙门氏菌Real-time PCR 扩增沙门氏菌图二 热贴扩增沙门14028菌株 DNA 的整个过程的温度监测。LAMP共反应70min.图一 应用热贴提供温度进行LAMP反应的结果盒大小对热贴温度的影响图三 RT-65 相变材料在煮沸时间不同的条件下，停止加热后维持温度的情况。图四 RT-62 相变材料在煮沸时间不同的条件下，停止加热后维持温度的情况。图五 RT-65 相变材料稳定性图六 不同量的CaO和H2O所产温度PMA控制控制LAMP污染污染实验过程中严格分区，提前配好所有反应管以减小污染PMA处理的LAMP产物浓度的测定PMA抑制效果的检测方法-跑胶及real-time LAMP无电加热完无电加热完成成LAMP反应反应反应室的设计相变材料稳定性多重多重LAMP-LFD反应体系的建反应体系的建立立胶体金制作方法的优化：煮沸时间；确切的试剂添加比例LFD双检测线方法的建立LFD双金垫的研发，从而增加检测灵敏度多重LAMP反应体系的建立快捷、简便快捷、简便多重致病菌多重致病菌的检测的检测