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1、实验实验三三、动物性食品生物性污染、动物性食品生物性污染 物的检验物的检验动物性食品卫生学实验动物性食品卫生学实验Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.一一. .生物性污染的来源生物性污染的来源p微生物污染微生物污染 (microorganism (microorganism pollutionpollution ) )p寄生虫污染寄生虫污染 (parasitical pollution ) (parasi
2、tical pollution )p昆虫污染昆虫污染 (insectical pollution ) (insectical pollution )Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Lt
3、d.内源性污染内源性污染外源性污染外源性污染外源性生物性污染是动物性食品微生物污染的主要途径外源性生物性污染是动物性食品微生物污染的主要途径Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.二二. .生物性污染的评价指标生物性污染的评价指标菌落总数菌落总数细菌总数细菌总数大肠菌群值大肠菌群值(MPN)致病菌检测致病菌检测(PCR法法)其他其他Evaluation only.Created with Aspose.Sli
4、des for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.实验目的实验目的掌握动物性食品菌落总数、大肠菌群测定掌握动物性食品菌落总数、大肠菌群测定及常见致病菌及常见致病菌PCRPCR检测的原理、步骤和方法检测的原理、步骤和方法了解动物性食品菌落总数的卫生标准,进了解动物性食品菌落总数的卫生标准,进行卫生判定行卫生判定Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2
5、011 Aspose Pty Ltd. 1. 1.鲜乳的微生物学检验鲜乳的微生物学检验菌落总数的测定菌落总数的测定检检 验验 处处 理理24 2h或或48 2h36 1 报报 告告菌落记数菌落记数每皿加入适量营养琼脂每皿加入适量营养琼脂选择选择3个适宜稀释度,吸取个适宜稀释度,吸取1ml加入灭菌平皿加入灭菌平皿作成几个倍数的稀释液作成几个倍数的稀释液Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.注注 意意 事事 项
6、项无菌操作无菌操作稀释度的选择稀释度的选择平均菌落数在平均菌落数在30-30030-300之间的稀释度为宜;之间的稀释度为宜;每个稀释度作两个平皿每个稀释度作两个平皿记数要求记数要求30-30030-300300300 30 30300300或或 30 30Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.2.2.鲜乳大肠菌群值的测定鲜乳大肠菌群值的测定Evaluation only.Created with Aspo
7、se.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.报报 告告根据证实为大肠菌群阳性的管数,查根据证实为大肠菌群阳性的管数,查MPNMPN检索表,报告检索表,报告每每100mL(g)100mL(g)检样中大肠菌群的最可能数。检样中大肠菌群的最可能数。判定标准判定标准Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Lt
8、d.3. 常见致病菌的快速检测常见致病菌的快速检测 SE的的PCR检测检测原原 理理 以扩增的以扩增的DNADNA分子为模板,以一对分别与模板分子为模板,以一对分别与模板互补的寡核苷酸片段为引物,在互补的寡核苷酸片段为引物,在DNADNA聚合酶的作用聚合酶的作用下,按半保留复制的机制沿着模板链延伸直至完下,按半保留复制的机制沿着模板链延伸直至完成新的成新的DNADNA合成。不断重复这一过程,可使目的合成。不断重复这一过程,可使目的DNADNA片段得到扩增。因为新合成的片段得到扩增。因为新合成的DNADNA也可以作为也可以作为模板,因而模板,因而PCRPCR可使可使DNADNA的合成量呈指数增长
9、。的合成量呈指数增长。Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.PCRPCR的基本成分的基本成分模板模板DNADNA特异性引物特异性引物(SEF14)(SEF14)热稳定热稳定DNADNA聚合酶聚合酶dNTPdNTP二价阳离子二价阳离子缓冲液缓冲液过过 程程( (高温变性、低温退火、中温延伸高温变性、低温退火、中温延伸) )Evaluation only.Created with Aspose.Slides f
10、or .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.引物设计引物设计 利用引物设计软件利用引物设计软件Primer5.0Primer5.0针对针对SEF14SEF14主要主要结构亚单位结构亚单位sefAsefA的基因序列设计引物的基因序列设计引物: : 上游引物上游引物Usef AUsef A为为5-5-ATGCGTAAATCAGCATCTGATGCGTAAATCAGCATCTG-3-3;下游引;下游引物物LsefALsefA为为5-5-TTAGTTTTGATACTGCTGAACTTAGTTTTGATACT
11、GCTGAAC-3-3Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.样品处理(模板制备)样品处理(模板制备)以以12000rpm12000rpm离心离心5min,5min,上清液即含基因组上清液即含基因组DNADNA待检肉样乳待检肉样乳1g/mL1g/mL以以9mL9mL增菌液混合研碎,增菌液混合研碎,制成食品匀浆液制成食品匀浆液3737振荡培养振荡培养3 3小时小时取样液取样液1mL1mL以以2000rpm200
12、0rpm离心离心2min,2min,取上清液取上清液以以8000rpm8000rpm离心离心5min,5min,沉淀以沉淀以50uL50uL悬浮悬浮沸水浴沸水浴10min10min后迅后迅速置冰浴速置冰浴5min5minEvaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.反应体系反应体系10Buffer 2.5uLdNTP 2.0MgCl2 2.0上游引物(上游引物(20uM) 1uL下游引物(下游引物( 20uM )
13、1uL模模 板板 2uLTaq DNA Polymerase 0.25 灭菌菌ddH2O up to 25uLEvaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.反应参数反应参数1. Initial denature 95 5 min 2. Denature 95 60s3. Anealing 47 60s 4. Elongation 72 60sReturn to 2 for 30 cycle5. Final elon
14、gation 72 10 minKeep 4 5 minEvaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.琼脂糖凝胶电泳琼脂糖凝胶电泳电泳条件:电泳条件:argrose 1% U 80V T 50minPCR产物产物+Loading buffer琼脂糖凝胶电泳琼脂糖凝胶电泳分析结果分析结果Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd. E-mail: Mobile-phone: 15823280210Evaluation only.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Copyright 2004-2011 Aspose Pty Ltd.