耐热菌的竞争定量pcr检测方法优化与建立

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1、中国农业科学2006,39(2):375-380ScientiaAgriculturaSinica耐热菌的竞争定量PCR检测方法优化与建立121李建科，冯再平，仇农学12(陕西师范大学食品工程系，西安710062；兰州理工大学生命科学与工程学院，兰州730050)摘要：【目的】研究苹果浓缩汁中耐热菌（Alicyclobacillusacidoterrestris）的定量PCR快速检测法。【方法】通过引物设计、PCR扩增及凝胶纯化试剂盒回收，构建并获得耐热菌的PCR竞争模板；用获得的竞争模板作为定量内标物建立耐热菌的竞争定量PCR（QC-PC

2、R）检测体系。【结果】经对建立的QC-PCR检测体系优化，目标4模板检测灵敏度由5×10个分子/PCR体系，提高到50个分子/PCR体系，竞争模板和目标模板分子共扩增可检测23到5×10个目标模板分子/PCR体系，并能从人工回添苹果浓缩汁样品中定量检测到5×10cfu/PCR体系的耐热菌，整个检测时间为4～5h，比传统的细菌培养皿培养记数法时间（4～5d）大大缩短。【结论】本研究建立的耐热菌竞争定量PCR检测法特异、快速，可作为微生物PCR竞争模板构建和建立竞争定量PCR的方法学参考，也可用于商品果汁和苹果浓缩汁工业化生产中耐热菌的快速检测

3、和质量安全控制。关键词：耐热菌；苹果浓缩汁；竞争定量PCROptimizationandEstablishmentofQuantitativelyCompetitivePCRSystemfortheDetectionofAlicyclobacillusacidoterrestris121LIJian-ke,FENGZai-ping,QIUNong-xue(12DepartmentofFoodEngineering,ShaanxiNormalUniversity,Xi’an710062;CollegeofLifeScienceandEngin

4、eering,LanzhouUniversityofTechnology,Lanzhou730050)Abstract:【Objective】TostudythedetectivemethodofAlicyclobacillusacidoterrestrisinapplejuiceconcentrate(AJC)byquantitativelycompetitivepolymerasechainreaction(QC-PCR)system.【Method】TheQC-PCRsystemfordetectionofAlicyclobacill

5、usacidoterrestriswasestablishedthroughtheprimerdesign,PCRamplification,andreclaimofgelpurificationreagentboxtoconstructandobtainthecompetitivetemplatewhichwasusedasquantitativeinternalstandards.【Result】TheQC-PCR4systemwasoptimizedandestablishedinthisstudy,andthedetectivese

6、nsitivityofthetargettemplatehadgottenbetterfrom5×10to250molecules/PCRsystem.Asaresultofthis,5×10targettemplatemolecules/PCRsystemwasdetectedwhenthetwotemplates3co-amplified,and5×10cfu/PCRsystemofAlicyclobacillusacidoterrestrisinAJCwasdetected.Thedetectivetime(4hto5h)isrema

7、rkablyshortenedfromtraditionalmethod（4dor5d）usingplateculturecounting.【Conclusion】Themethodinthisstudyisbetterthanothersinefficiencyandspecificity,whichcanbeareferenceforcompetitivetemplateconstructionofmicrobePCRandconstructivemethodologyofQC-PCR.Ithaspotentialtobeapplied

8、inAJCcommercialproductionprocessfortherapiddetectionofAlicyclobacillusacidoterrestris.Key