美國維康公司真菌毒素分析技術

AflaTestforPeanuts

測定花生中的黃曲霉毒素的維康技術AflaTest法OutlineAflaTestProcedure

分析操作步驟Extraction:Sample+Methanol

Blend

Filter(paper)提?。簶悠?甲醇/水，混勻，過濾（濾紙）Dilution:Extract+dH2O

Filter(microfiber) 稀釋：提取液+水，過濾（玻璃纖維濾紙）Chromatograph:sample

AflaTestcolumn. 色譜操作：樣品通過親和柱Wash:withdH2O清洗：用水清洗Elution:Methanol

AflaTestcolumn

Aflatoxin

洗脫：用甲醇將親和柱上的黃曲霉毒素洗脫下來Fluorometer熒光計法 HPLCHPLC法1.AddDeveloper加衍生液水 1.AdddH2O加2.ReadonFluorometer 2.InjectonHPLC在熒光計上讀數(shù)注入HPLC儀中Setup實驗準備Prepareextractionsolution(60%Methanol/H2O)everyweekorasneeded.制備提取液（甲醇60：水40Calibratefluorometer標定熒光計Red紅色=44,Green綠色=-1,Yellow黃色=22±2Makesurethat2mlofpurifiedwaterreads0ppb 確認2ml純水，讀數(shù)為0PrepareAflaTestdevelopersolutiondailyorevery8hours(5mldeveloper+45mlpurifiedH2O)制備衍生液（一天配一次）（衍生液原液1：純水9）Makesurethereagentblank(1mlMethanol+1mldeveloper)reads0ppb 確認試劑空白（1ml甲醇+1ml衍生液）讀數(shù)為0SampleExtraction樣品的提取Weigh25ggroundpeanuts(raworroasted),peanutmeal,orpeanutbutter.稱取25克磨細的花生樣品（粗花生、烤花生、花生仁、花生醬）Add5gsalttosample.加5克鹽Placesampleinblenderjarandadd125mls60%Methanol/H2O.將樣品+鹽置于攪拌杯中，加125ml的甲醇/水Coverblenderjarandblendathighspeedfor1minute.蓋上攪拌杯蓋，高速攪拌1分鐘Pourextractintoflutedfilterpaperandcollectfiltrateinacleanvessel

取下蓋子，將提取物到入折疊式濾紙上，濾液收集于干凈的容器中。

ExtractDilution提取物的稀釋

Pipetorpour20mlfilteredextractintoacleancontainer.

移取20mL上步的濾液，置于干凈的容器中。

Diluteextractwith20mlsdistilledwater.Mixwell.用20mL純水將濾液稀釋，混勻。Filterdilutedextractthroughglassmicrofiberfilter. 將上步稀釋液通過玻璃纖維濾紙。ColumnSeparation親和柱分離操作AttachAflaTest-Pcolumntoglasssyringebarrelreservoironpumpstand. 將親和柱與注射器筒相連Measure5mlsoffilteredextractintothereservoirandpushtheentireextractslowlythroughcolumn.將5ml濾液加到注射器筒中，使其慢慢地通過親和柱Measure10mlsdistilledwaterintothereservoirandpushthroughcolumn.用10ml純水清洗注射器筒和親和柱ElutionofAflatoxinsfromColumn洗脫親和柱上的黃曲霉毒素Addexactly1mlHPLCgrademethanoltoreservoirandslowlypushthroughcolumn.Collectalleluateinglasscuvette.加入1ml色譜級的甲醇，使其慢慢地通過親和柱，洗脫液置于測試管中。Addexactly1mldiluteAflaTestdevelopertoeluateincuvette.Mixwell.將1ml衍生液加入到測試管的洗脫液中。Placecuvetteinacalibratedfluorometerimmediately.立即將測試管置于已標定好的熒光計中Recordreadoutinppbafter60seconds60秒后記錄測定結果

SummaryAflaTestProcedureforPeanuts測定花生中黃曲霉毒素分析方法一覽表Calculationofsamplegramequivalents

相當樣品重量數(shù)的計算方法25gsample 15ml X X15ml=1.0g125mL 30ml25gsample 20ml X X5ml=0.5g125mL 40mlAmt.sampleVol.ext.solutionXVol.ext.usedindilutionTotalVol.ofdiluext.XVol.passcolumn=GramequivalentAflaTestColumnCapacity

黃曲霉毒素親和柱的柱容量計算100ngtotalaflatoxin(B1,B2,G1,G2)100納克黃曲霉毒素總量(B1+B2+G1+G2) 0-100ppb 1.0gsampleequivalent 0-200ppb 0.5gsampleequivalent 0-500ppb 0.2gsampleequivalent*1ppb=1ngofaflatoxin/gramofsample =1μgofaflatoxin/kgofsample1ppb=1納克黃曲霉毒素/1克樣品=1微克黃曲霉毒素/1公斤樣品

TheHighestPossibleReadingforAflatoxin?測定結果讀數(shù)的解釋AflaTestPColumnsholdatleast100ngoftotalaflatoxinsAflaTestP親和柱對黃曲霉毒素總量的容量大于100ngThiscorrespondsto對應于100ppbfora1gsampleequivalent(usually10ml)100ppb:1g樣品(10ml濾液)200ppbfora0.5gsample(5ml).200ppb:0.5g樣品(5ml濾液)Wehaveobtainedreadingsashighas800ppb.曾經(jīng)獲得過800ppb的讀數(shù)Thisnumberisnotaccurate,butdoesindicateasamplecontainingatleast800ppbaflatoxinifthesamplewasruncorrectly.800ppb的讀數(shù)是不準確的，但它表示已經(jīng)超過了800ppb。Samplesreadinghigherthanthecolumncapacityshouldbererunwithlesssampleoverthecolumnandtheresultmultipliedaccordingly.如果結果讀數(shù)超過了柱容量，可以減少通過親和柱的濾液量，重新試驗，測定結果根據(jù)減少的倍數(shù)計算。Forexample,forthe0.5g(5mlmethod),ratherthanpassing5mLoverthecolumn,pass1mloverthecolumnandmultiplytheresultby5.例如：對于0.5g的方法（用5ml濾液），不用5ml濾液，而是用1ml濾液通過親和柱，這樣測定結果乘以5。TROUBLESHOOTINGAflaTest

黃曲霉毒素分析方法可能會遇到一些問題CheckReagentsdaily.每天檢查試劑Makesuremethanol,H2O,andmethanol+developershouldreadzeroorlessonthefluorometer.確認甲醇、純水，甲醇+衍生液的讀數(shù)等于或小于0Avoidcontactofreagentswithsoftflexibleplasticorrubber.避免試劑與軟塑料、軟橡膠接觸Methanolandwatershouldbestoredinglasscontainers.甲醇和水應該儲存在玻璃容器中。Makesuretoaddsalttosample.確定樣品中加入了鹽Makesuresalthasnoadditives.確定鹽中沒有雜質Problem:Falsehighreadings讀數(shù)高

Becarefulnottotearmicrofiberfilter.不要弄破了玻璃纖維濾紙Makesuresampleisclearaftersecondfilter.確認第二次過濾后，濾液是否清亮Placesampleoncolumnimmediately.立即將樣品濾液通過親和柱UseequipmentspecifiedbyVicam,esp.cuvettes.確保使用維康公司推薦的產(chǎn)品，比如測試管Usecleancuvettes.使用干凈的測試管Checkforacontaminantsinsidethecuvette檢查測試管內(nèi)壁是否有污染物Checkdirtorfingerprintsontheoutsideofthecuvette.檢查測試管外壁上是否有手指印和污物WipecuvettewithKim-wipebeforeputtingintofluorometer.用擦拭紙擦凈測試管

Donotmixcuvettebyputtingthumbover

topofcuvetteandshaking振蕩測試管時，不要將手指置于測試管的頂部Makedeveloperfreshdailyfromconcentrate.Donotusedilutedeveloperifovereighthoursold.衍生液需要當天配制，不要用超過8小時以上的衍生液Donotuseexpiredconcentrate.不要用過期的原裝濃衍生液KeepdeveloperinAMBERbottle,protectfromlight.

確認衍生液置于安培瓶中，避光。Typesofsamplesnotlistedinthemanualmaynotyieldoptimalresults.操作手冊中未列入的樣品，可能不會得到最好結果。

Problem:Falselowreadings讀數(shù)低

Passsamplethroughcolumnslowlyandelutesamplefromcolumnslowly(1-2drops/second). 樣品通過親和柱和從親和柱上洗脫黃曲霉毒素的速度要慢（1-2滴/秒）Checktomakesuremethodisfollowedcorrectly.Allmethodsshouldusetwofiltrations. 檢查操作過程不要漏掉任何步驟，所有方法中，必須進行兩次過濾。Makesure60%methanolextractionsolutionismadecorrectlyandislessthanoneweekold.

確定所用的甲醇提取液的甲醇含量為60%，而且配制時間不能超過一周。Problem:Readingsareinconsistent問題：讀數(shù)有變化Besuretocomparereadings確信在下列情況下進行的讀數(shù)比較fromthesamesamplefiltraterunatthesametime用同一樣品的濾液做的平行實驗orfromsamplesmixedverywell.樣品混合的非常好。Differentsamplescangivevariationsinreadings.不同的樣品得出不同的讀數(shù)。Followinstructionscarefully.仔細地按照操作手冊做實驗Runasamplefromstarttofinishwithoutstopping.

對一個樣品進行檢測時應該是從開始到結束是不間斷的Calibratefluorometercorrectlyfortheprocedureyouareusing.根據(jù)你所用的方法正確標定熒光計。Keepstandardsawayfromlightandreplacestandardseveryyear.標準物要避光保存同時每年要更換一次。

Mixfiltratewellafterdiluting.稀釋后濾液應充分混合Collectallofthesampleeluateinthecuvette收集全部的樣品洗脫液在試管中Makesuredispensingtubesareprimedandfreeofairbubbles確信所用的測試管沒有質量問題，沒有空氣泡Discardfirstsquirtfrommethanolanddeveloperbottles.從甲醇和衍生液分配器中第一次流出的液體必須丟棄Afteraddingdeveloper,mixwell,andplaceintofluorometerimmediately.加入衍生液后，混合均勻，然后立即放入熒光計進行測量。Mixcuvettewellafteraddingdeveloper加入衍生液后必須混合均勻。Usea60secondtimedelay應使用60秒的時間延遲Runachecksampledaily每天做一個比對樣品的檢查CommonSeries4FluorometerProblems4系列熒光計的常見問題1. Problem:Resultsvaryfrom0to270ppbonthesamesample.問題：對同一個樣品，檢測結果從0-270ppb不等。Besuretopushthestandardvialsandcuvettesfullyintotheinstrumentsothatthebottomofthevialtouchesthebottomofthesamplewell.

確定標準物小瓶和測試管完全插入到儀器的底部，使小瓶能同樣品池的底部接觸，。2.Problem:Itishardtopressthenumbersonthekeypad.問題：鍵盤上的數(shù)字鍵難于按動。ReplacementkeypadandinstructionsonhowtochangethekeypadareavailablefromVicam.更換鍵盤，關于如何更換請與VICAM聯(lián)系。3.Problem:DataError:問題：數(shù)據(jù)錯誤Makesurefinaleluateincuvetteisnotcloudyorstronglycolored.確定在測試管中的最終洗脫液沒有渾濁現(xiàn)象也沒有較深的顏色。4. Problem:Displayproblem問題：顯示問題Clearmemoryandsetthedisplayusingtheprocedure(onPage93)清除內(nèi)存重新設置顯示(參看93頁)5.Problem:CanSeries4fluorometerbeleftonovernight?問題：4系列熒光計能過夜關機行嗎？Yes,butturninstrumentoffattheendofthedayifyouexpectcurrentfluctuations.是，如果你感到電源電壓有波動，在每天下班時，你可以將儀器后面的電源開關關掉。Fluorometercanberecalibratedailybypressingoptionskey用options鍵，可以每天對熒光計進行標定1.

AttheVICAMREADY”typethisnumbersequence:8,3,1,1,5.在“VICAMREADY”狀況下，輸入8,3,1,1,5。2.Thedisplaywillshow“CLEARMEMORY?”,pressENTER.顯示“CLEARMEMORY?”，按ENTER鍵。3.Thedisplaywillshow“CONFIRMCLEAR?”,press1.Thememoryisnowcleared.顯示“CONFIRMCLEAR?”，按1，此時存儲器已被清理。4.Atthe“VICAMREADY”displaypressthesenumbersonceeachtime:7,5,7,6,1,2.Youwon’tseeanychangeinthedisplay“VICAMREADY”.在“VICAMREADY”狀況下，逐次按“7,5,7,6,1,2”。此時，你不會看到任何變化。5.Forinstrumentswithserialnumbersgreaterthan177,pressthenumber2.對于系列號大于177的儀器，按2。6.Thiswillsetthedisplaytothelatestrevision.Afterthat,testtheinstrumentfornormaloperation.此時，顯示的是最后一次修改版本，儀器可以重新使用。Clearmemoryandsetthedisplay

清理存儲器、設置顯示Keeproomtemperatureconsistent.一直要保持室溫Gentlyblowoutcuvetteholderwithcannedair.用瓶裝空氣輕輕地吹洗測試管支架Readyellowvialimmediatelyaftercalibration,thenperiodicallyduringtheday.標定后立即測定黃色驗證管，在一天的測定工作中，時常用黃色驗證管檢查。Calibrationvialsshouldbeclearwithnofloatingparticles.標定管要干凈，不要吸附任何雜質。Ifvalueshiftssignificantlyovertime,everyday,whentheroomtemperatureisconsistent,thefluorometerneedstobesentbackforrepair.如果室溫沒有變化，讀數(shù)還是經(jīng)常漂移，請將熒光計送回廠家修理。6.Problem:Fluorometerisnotholdingcalibration問題6：熒光計不能讀入標定值Keepinstrumentprotectedfromdustandparticles.儀器要防止灰塵Gentlyblowoutcuvetteholderwithcannedair.用瓶裝空氣輕輕地吹洗測試管支架InstrumentcanbesenttoVicamforroutinecheckandcleaning.可以將送回Vicam做保養(yǎng)PleasecallCloverorVICAMbeforesendingafluorometerforrepairorcheck.儀器需要修理或保養(yǎng)時，請預先與祺照或維康公司聯(lián)系WhatcanIdoforperiodicmaintenance?

需要做那些經(jīng)常性的保養(yǎng)AflaTestforMilk

測定牛奶中黃曲霉毒素分析方法AflaTestMilkProcedure操作步驟Centrifuge50mlmilksamplewith1gsaltat2000gfor10minutes 在50ml牛奶樣品中加入1g鹽，在2000g條件下，離心10分鐘。Removeskimportionofmilk(bottomlayer) 將牛奶浮皮（底部）除掉Filterskimmilkthroughmicrofiber. 將脫脂牛奶用玻璃纖微濾紙過濾Load10mlskimmilkontoAflaTestcolumn. 將10ml脫脂牛奶濾液通過親和柱AflaTestMilkProcedure操作步驟Wash:with10%Methanol/H2Otwice. 用10%甲醇/水洗兩次Elute:with1ml80%Methanol/H2O. 用1ml80%甲醇/水洗脫Add1mlAflaTestdevelopertoeluate. Mix.加1ml衍生液，混勻Placecuvetteincalibratedfluorometer. 將測試管置于熒光計中TroubleshootingAflaTestMilk

測定牛奶時可能會碰到的問題Problem1.Unabletopushmilkthroughcolumn牛奶無法通過親和柱Wholemilkneedstobecentrifuged全奶需要離心分離thebottomlayermustbetakenwithoutdisturbingthetopfatlayer.取走底部層，不要攪爛頂部的脂肪層Centrifugeat2000gfor10to15minutes.(Thismaybe5000rpm)在2000g條件下離心10至15分鐘（轉速可能為5000轉/分鐘）Don‘tforgettoaddsaltandfiltersamplethroughmicrofiberfiltersample.不要忘記加鹽、用玻璃纖微濾紙過濾Milkisbestrunatroomtemperature.牛奶最好在室溫下分析Problem2.Falsepositives問題2假陽性Useacleansyringebarrelforwashingandelution用干凈的注射器筒進行清洗和洗脫Placepartoffirstwashdirectlyintocolumnheadspace.將第一次洗液直接接入親和柱中Elutewith80%Methanol,not100%Methanol.用80%甲醇，而不是用100%甲醇進行洗脫Problem3.Falsenegatives問題3假陰性CalibratefluorometerwithAflaTestMilkstandards 用AflaTest-M標定物標定熒光計MakesuretousecorrectpercentMethanol/H2Osolutionforwashing(10%Methanol)andelution(80%Methanol)確認洗液（10%甲醇）和洗脫液（80%甲醇）中甲醇/水的比例是否正確AflaTest,OchraTest,FumoniTestandZearalaTestforCorn

測定玉米中的黃曲霉毒素、赭曲霉毒素、伏馬毒素、玉米赤霉烯酮的分析方法OchraTest赭曲霉毒素分析方法50ggroundsample+5gsalt+100ml80%Methanol/H2O.50g磨細樣品+100ml80%甲醇/水Blendoneminuteandfilter.攪拌1分鐘、過濾Dilute10mLextractwith40mLwater.用40mL水稀釋10mL的濾液Filterthroughmicrofiberfilterpaper.用玻璃纖微濾紙過濾Pass10mLextractthroughOchraTestcolumn.將10mL濾液通過OchraTest親和柱Washcolumnwith10mLMycotoxinWashBuffer.用10mL真菌毒素緩沖液洗