Improved Method for Purification of Bacterial DNA from Bovine Milk for Detection of Brucella spp.docx
- 文档编号:11518095
- 上传时间:2023-06-01
- 格式:DOCX
- 页数:11
- 大小:68.02KB
Improved Method for Purification of Bacterial DNA from Bovine Milk for Detection of Brucella spp.docx
《Improved Method for Purification of Bacterial DNA from Bovine Milk for Detection of Brucella spp.docx》由会员分享,可在线阅读,更多相关《Improved Method for Purification of Bacterial DNA from Bovine Milk for Detection of Brucella spp.docx(11页珍藏版)》请在冰点文库上搜索。
ImprovedMethodforPurificationofBacterialDNAfromBovineMilkforDetectionofBrucellaspp
AppliedandEnvironmentalMicrobiology,August1999,p.3735-3737,Vol.65,No.8
0099-2240/99/$04.00+0
Copyright©1999,AmericanSocietyforMicrobiology.Allrightsreserved.
ImprovedMethodforPurificationofBacterialDNAfromBovineMilkforDetectionofBrucellaspp.byPCR
C.RomeroandI.Lopez-Goñi*
DepartamentodeMicrobiología,UniversidaddeNavarra,31008 Pamplona,Spain
Received4February1999/Accepted9May1999
ABSTRACT
Top
Abstract
Text
References
DifferentmethodsofextractionofbacterialDNAfrombovinemilktoimprovethedirectdetectionofBrucellabyPCRwereevaluated.WefoundthattheuseofalysisbufferwithhighconcentrationsofTris,EDTA,andNaCl,highconcentrationsofsodiumdodecylsulfateandproteinaseK,andhightemperaturesofincubationwasnecessaryfortheefficientextractionofBrucellaDNA.ThelimitofdetectionbyPCRwas5 to50 BrucellaCFU/mlofmilk.
TEXT
Top
Abstract
Text
References
Brucellaspp.aregram-negativebacteriawhichcausebrucellosis,awidespreadzoonosis.Theeconomicimportanceofbrucellosisrequirestheuseofsensitiveandrapiddiagnosismethods.Atpresent,diagnosisofbrucellosisinlivedairycattleinvolveeithertheisolationofBrucellafrommilksamplesorthedetectionofanti-Brucellaantibodiesinserumormilk
(1).However,thesemethodsarenotwhollysatisfactory.Bacteriologicalisolationisatime-consumingprocedure,andhandlingthemicroorganismishazardous.Serologicalmethodsarenotconclusive,becausenotallinfectedanimalsproducesignificantlevelsofantibodiesandbecausecross-reactionswithotherbacteriacangivefalse-negativeresults
(1).SomepreviousstudieshavedemonstratedthatPCRcanbeusedtodetectBrucellaDNAinmilksamples(4,7,10,12).PCR-basedmethodshavethepotentialtobefast,accurate,andefficientindetectingBrucella.However,whenPCRwasappliedtomilksamples,itssensitivitywaslowwithrespecttobacterialculture,andsomefalse-negativePCRresultshavebeenreported(10).Thedifficultyassociatedwithlysingthemicroorganismscouldaccount,atleastinpart,forthefailureofthePCRassayinsamplesthatwereculturepositive.Todealwiththisproblem,wecompareddifferentmethodsofextractionofbacterialDNAfrombovinemilktoimprovethedirectdetectionofBrucellabyPCR.Theresultsaredescribedinthispaper.
SterilebovinemilkwasinoculatedwithBrucellaabortus2308 to2 × 105CFU/ml,andserialdilutionswerepreparedinmilktodeterminethelimitofdetection(expressedasCFUpermilliliter)ofthePCR.DifferentmodificationsoftheDNAextractionmethodpreviouslydescribed(10)wereused.Frozenmilkwasthawedatroomtemperature,and500 µlofsamplewasmixedwith100 µlofTEbuffer(1 mMEDTA,10 mMTris-HCl[pH7.6])orNETbuffer(50 mMNaCl,125 mMEDTA,50 mMTris-HCl[pH7.6]).Differentcombinationsofdenaturingagentswereadded:
50 µlof2.6 NNaOHsolution,100 µlof24%sodiumdodecylsulfate(SDS)(finalconcentration,3.4%),or100 µlof10%Zwittergent3-14 detergent(Zw3-14 [Calbiochem-BehringCorp.];finalconcentration,1.4%).Themixturewascooledoniceafterincubationatroomtemperatureor80 or100°Cfor10 min.Differentcombinationsofenzymaticconditionsweretested:
proteinaseK(SigmaChemicalCo.;finalconcentration,162, 325, or650 µg/ml)at37 or50°Cfor0.5, 1, 1.5, 2, 2.5, or3 h;lysozyme(Sigma;finalconcentration,162, 325, 650, 1,300, or2,600 µg/ml)at37°Cfor1 h;orRNase(ICNPharmaceuticalsInc.;finalconcentration,19, 37, 75, 150, or300 µg/ml)at50°Cfor0.25, 0.5, 1, 1.5, or2 h.Insomeexperiments,celldebriswereremovedbyprecipitationwith5 MNaClandhexadecyltrimethylammoniumbromide-NaCl(CTAB-NaCl)solutionat65°Cfor10 min(13).DNAwasextractedbystandardmethodswithphenol-chloroform-isoamylalcohol,precipitatedwithisopropanol,washedwithethanol,anddriedundervacuum(11).TheDNApelletwasdissolvedin25 µlofsteriledistilledwaterandstoredat
20°Cuntilfurtheruse.A1-µlvolumeofthisDNAsolutionwasaddedtothePCRcocktail.Alternatively,DNAwasextractedfromthemixtureaftertheincubationwithproteinaseKandRNasebyusingtheInstagene(Bio-RadLaboratories)orthePrep-A-Gene(Bio-RadLaboratories)systemasspecifiedbythemanufacturer.AfinalpurificationstepwithSephacrylS-300orS-500(PharmaciaBiotech)wasalsoassayed.Atotalof25 µlofpurifiedDNAwasaddedto200 µlofa50%(vol/vol)solutionofSephacrylS-300orS-500indistilledwater,andthemixturewasincubatedatroomtemperaturefor10 min.Aftercentrifugation(13,000 × gfor5 min),thesupernatantwasusedforPCR.Inallexperiments,onesampleofsterilemilkwasincludedasinternalnegativecontrol.AmplificationanddetectionofBrucellaDNAbyPCRwasperformedwithprimersF4andR2asdescribedpreviously(9,10).InallPCRassays,apositivecontrol(B. abortus2308 DNA)andanegativecontrol(sterilewater)wereincluded.Generallyrecommendedprocedureswereusedtoavoidcontamination(8).
Theeffectsoftemperatureandthetypeofdenaturingtreatment(SDSorZw3-14 detergentsinNETorTEbuffer)onthePCRresultswerestudied.Intheseexperiments,theextractionofDNAwasfollowedbydigestionwithproteinaseK(325 µg/mlat50°Cfor2 h)withoutRNasetreatment.ApositivePCRresultwasobtainedonlywhentheDNAextractionwasperformedwithSDSinNETbuffer(Fig.1),andmorereproducibleamplificationswereachievedwhenthesamplewasincubatedat80°C.TheeffectofNaOHasadenaturingagentwasalsotestedinNETbufferwithorwithoutSDS.TheamplificationinthepresenceofNaOHalwaysresultedinfainterbands(Fig.1).Inaddition,digestionwithlysozymedidnotimprovetheamplificationevenatthehighestconcentrationtested(datanotshown).Therefore,allsubsequentDNAextractionswereperformedwithNETbufferandSDSat80°C.
Viewlargerversion(108K):
[inthiswindow]
[inanewwindow]
FIG.1. EffectoflysisbuffercompositionanddenaturingagentonthedetectionofBrucellaDNAbyPCR.Samplesinlanes2 to7 weresterilebovinemilkinoculatedwithB. abortus(2 × 105CFU/ml).Lanes:
1, negativecontrolwithoutDNA;2 and3, DNAextractedwithSDSandTEbuffer;4 and5, DNAextractedwithSDSandNETbuffer;6 and7, DNAextractedwithSDS,NaOH,andNETbuffer;8, positivecontrolwithB. abortusDNA;9,
X174DNA/HaeIIImarker(BoehringerMannheim).Thelysisincubationtemperaturewas80°Cinlanes2, 4, and6, and100°Cinlanes3, 5, and7. Thesizeoftheamplificationproductisabout905 bp.NoamplificationwasdetectedwhenZw3-14 wasusedinsteadofSDS(datanotshown).
TheeffectsofthetreatmentwithproteinaseKandRNaseatvariousconcentrationsonthePCRresultswerealsostudied.NodifferenceswerefoundwhenproteinaseKwasaddedtoafinalconcentrationof325 or650 µg/ml,buttheamplificationwasweakwhensmalleramountsoftheenzymewereadded(datanotshown).Thebestresultswereobtainedwhentheincubationwascarriedoutforatleast1.5 h.Incubationtemperaturesof37 and50°Cdidnotgivedifferentresults.Similarexperimentswererepeated,includinganRNaseincubationsteppriortotreatmentwithproteinaseK. Astrongerandmorereproducibleamplificationwasachievedwhenthesamplewasincubatedwith75 µgofRNasepermlat50°Cfor2 h(datanotshown).Increasingtheenzymeconcentrationfurtherdidnotchangetheefficiencyoftheamplification.Therefore,allsubsequentexperimentsincludeddigestionwithRNase(75 µg/ml)followedbyincubationwithproteinaseK(325 µg/ml),bothat50°C.
TheeffectofremovalofcelldebrisbyprecipitationwithCTAB-NaClonPCRperformancewasalsotested.Ourresultsdemonstratedthatthistreatmentwasnotcritical(datanotshown).Inaddition,toavoidexcessivemanipulationofthesample,thepossibilityofreplacingthestandardDNAextractionmethodbycommercialsystemswasstudied.WhentheInstagenesystemwasusedtheamplificationwasalwaysweaker.However,theamplificationsignalobtainedwiththePrep-A-Genesystemwassimilartotheoneobtainedwiththestandardmethod(Fig.2),buttheresultswerelessreproducible.ToremovepossiblePCRinhibitorspresentintheDNA,afinalpurificationstepwithSephacrylS-300orS-500wasalsotested.TheresultsofthePCRobtainedafterthesetreatmentswerealwaysnegative(datanotshown).
Viewlargerversion(109K):
[inthiswindow]
[inanewwindow]
FIG.2. EffectoftheDNAextractionwithcommercialsystemsonthedetectionofBrucellaDNAbyPCR.Samplesinlanes3 to5 weresterilebovinemilkinoculatedwithB. abortus(2 × 105CFU/ml).Lanes:
1,
X174DNA/HaeIIImarker(BoehringerMannheim);2, positivecontrolwithB. abortusDNA;3, DNAextractedwiththePrep-A-Genesystem;4, DNAextractedwiththeInstagenesystem;5, DNAextractedwithphenol-chloroform-isoamylalcohol;6, negativecontrolwithoutDNA.
WealsodeterminedthelimitofPCRdetectionofBrucellaDNApurifiedbytheoptimizedmethodunderourconditions(NETbuffer,SDSat80°C,digestionwithRNaseandproteinaseKat50°C,andorganicextraction).SterilebovinemilkwasinoculatedwithaknownconcentrationofBrucellaandsubsequentlyprocessedforPCRamplificationandculture.ApositivePCRresultwasalwaysobtainedwithdifferentaliquotscontainingatleast50 CFU/mlofmilk(Fig.3).However,theamplificationsignalwasobtainedinonly50%ofthealiquotscontaining5 CFU/mlofmilk.
Viewlargerversion
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- Improved Method for Purification of Bacterial DNA from Bovine Milk Detection Brucella spp
![提示](https://static.bingdoc.com/images/bang_tan.gif)
链接地址:https://www.bingdoc.com/p-11518095.html