金黄色葡萄球菌coa基因实时荧光pcr的建立及应用研究

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1、金黄色葡萄球菌coa基因实时荧光PCR的建立及应用研究黄丰光乐燕红黄建城张秀莲河源市疾病预防控制中心R的建立金黄色葡萄球菌coa棊因TaqMan-MGB探针实时荧光PCR，并在食品安全风险监测中应用。方法根据金黄色葡萄球菌coa基因序列设计引物与TaqMan-MGB探针，建立并优化实时荧光PCR反应体系，并在食品安全风险监测中应用,同步进行传统细菌分离，比较2种检测方法的符合性。结果建立金黄色葡萄球菌coa基因TaqMan-MGB探针实时荧光PCR仅需42min可完成检测,与表皮葡萄球菌、链球菌等非目标细菌无交叉反应,对重组质粒的灵敏度达5copy/u1;对200

2、份食品安全风险监测标本进行快速筛斉，检测出15份阳性，传统方法也从相应样本中分离出15株金黄色葡萄球菌。结论针对金黄色葡萄球菌coa基因建立的TaqMan-MGB探针实时荧光PCR,可提高食品检测速度和灵敏度。关键词：金黄色葡萄球菌;实时荧光PCR;TaqMan探针；食品安全风险监测;凝固酶某0^Establishmentandstudyofreal-timefluorescencePCRforcoageneofStaphylococcusaureusHUANGFeng-guangLEYan-hongHUANGJian-chengZHANGXiu-lianHeyu

3、anMunicipalCenterforDiseaseControlandPrevention;Abstract：ObjectiveToestablishaTaqMan-Minorgroovebinder(MGB)probe-basedreal-timePCRmethodforcoageneofStaphylococcusaureus,andtostudyitsapplicationinthefoodsafetyriskmonitory.MethodsBasedontheconservativesequenceofcoageneofStaphylococcusau

4、reus,specificprimersandTaqMan-MGBprobesweredesigned,andtherapidreal-timePCRassaywasestablishedandoptimized.Itcouldbeeffectivelyappliedinthefoodsafetyriskmonitory.Thetraditionalmethodwasusedforisolatingbacteriasimultaneously,inordertocomparethecoherenceofthetwodetectionmethods.ResultsT

5、heTaqMan-MGBprobe-basedreal-timePCRassayforcoageneofStaphylococcusaureuscouldbefinishedin42minutes,andhadnocrossingreactionwithothernon-targetedbacteria,suchasepidermisStaphylococcus,Streptococcus.Thesensitivityoftherecombinantplasmidswas5copy/u1.200foodsafetyriskmonitoringspecimenswe

6、reconductedforrapidscreening,amongwhich15sampleswerepositive,and15samplesofStaphylococcusaureuswereisolatedbythetraditionalmethod.ConclusionThetechniqueofTaqMan-MGBprobe-basedreal-timePCRassayforcoageneofStaphylococcusaureuscanimprovethespeedandsensitivityoffooddetection.Keyword：Staph

7、ylococcusaureus;Real-timePCR;TaqMan-MGBbasedprobe;Foodsafetyriskmonitoring;Coagulasegene;金黄色葡萄球蘭是肉类、奶类等食品中污染常见的致病菌，产生的肠毒素能引起食物中毒U1，对食品安全造成较大的威胁。传统的分离培养方法通常需要3d~4dm，周期长、操作繁琐，给口益繁重的食品安全风险监测带来较大的压力。实时荧光PCR快速、特异、灵敏U1，在病原微生物检测中应用广泛。本研宄拟针对金黄色葡萄球菌coa基因建立TaqManMGB探针实时荧光PCR,并探讨在食品安全风险监测的应用效果。1

8、材料与方法