PCR技術(shù)檢測乳品中金黃色葡萄球菌研究.pdf

1、河北農(nóng)業(yè)大學(xué)碩士學(xué)位論文PCR技術(shù)檢測乳品中金黃色葡萄球菌研究姓名：楊洋申請學(xué)位級別：碩士專業(yè)：微生物學(xué)指導(dǎo)教師：張偉20050618StudyonPolymeraseChainReactionAssayforDetectionofStaphylococcusaureusinDairyProductsYangYmgMajor：Micwbiology(SchoolofFoodScience&TechnologyHdxiAglicultur

2、alUniversity)SI】碑m妍：PIID缸sor丑mgWeiAbstractStaphylococcusaureushasbeenconsideredoneofthemostimportantbacteriacausingfoodpoisoningIntheword，therearemanycasesthatarecausedbySaureusFoodsthatarefrequentlyincriminatedinstaphyl

3、ococcalfoodpoisoningincludemeatandmeatproducts，milkanddairyproductsandsoonTheconventionalmethodofinspectingfoodforpossiblecontaminationbycoagulasepositiveStaphylococcusaureusistootime—consuming，whichcantake3to5daystocomp

4、leteAnditisnotenoughsensitivetodetectSaureusInthisstudyspecificallyprimershasbeendesignedandarapidmethodhasbeenestablishedtodetectStaphylococcusaureusinfoodtoshortentimeofdetection，increasethesensitivityofmethodandprovid

5、eatechnicalhelpInthisstudyDNAconcentration，MgCl2concentration，，annealingtemperatureandPCRcirclesareoptimizedtodeterminetheoptimalPCRThereactionmixtureconsistedof5lof10xPCRamplificationbuffer(200mMnis—HCl[pH83】，500mMKCl，1

6、5mmMga2，01％[wt／vol】gelatin，05％Tween20)，4mofdNTPs(1mMeachinsolution)，05Plofeachprimer(40uMstocksolution)，025rqofTaq(5U塒一ofstocksolution)，2ⅢofDNAand3775mofdoubledistilledwaterThereactionwasrununderthefollowingconditions：Co

7、o】slarl；DNApre—denaluralional94℃for4min；DNAdenalurationat94℃for1rain，primerannealingat52℃for05rain，andDNAextensionat72℃for15minfor35cycles；thelastextensionwasperformedat72℃for35minThePCRproductswereexaminedbyelectrophore

8、siswith2％agarosegelSyntheticoligonucleotideprimersof21and24bases，respectivelywereusedinthepolymerascchainreactiontoamplifyasequenceofthenucgene，whichencodesthethermostablenucleaseofStaphylococcusaureusADNAfragmentof279bp

9、wasamplifiedPCRproductswereconfirmedbyDNAsequencingTherewere80bacterialstrainstobedetectedincluding60&aureusstrainsand20otherbacterialstrainsexceptforSaureusstrainsinordertodeterminespecificityofamplificationofprimersThe

10、resultsof60strainsofStaphyloCOCCUSaureuswerepositiveandthoseof20otherstrainswerenegativeWilhtheisolatedtargetthelowerdetectionlimitwas0675pgofDNAT1leeffectof6methodsofextractingDNAfromStaphylococcusaureusindairyproductsw

11、erecomparedAefficientextractionprocedurewasconfirmedforextractionofStaphylococcusaureusDNAfromdairyproductsStaphylococcusaureusDNAwasdirectlyextractedfromdairyproductswithoutenrichmentTheextractedDNAwassuitableforPCRdete

12、ctionAsolventextractionprocedurewasSuCCeSSfullymodifiedforextractionofStaphylococcusaureusDNAfromartificallycontaminatedwholemilk，skimmilkandcheeseThesensitivityoftheuniplexPCRis10cfuml～ofwholemilkskimmilkand55cfu91chees