【作者】 狄慧；

【导师】 张伟；

【作者基本信息】 河北农业大学 ， 农产品加工及贮藏工程， 2012， 硕士

【摘要】 食品安全突发事件频率高是近年来食品安全问题的一个显著特点,在各种食品安全事件中,病原菌的污染是引起食源性疾病的主要因素之一,快速检测食品中病原菌是及时有效预防病原菌传播及食品中毒的重要前提。常见的食源性致病菌主要有金黄色葡萄球菌、沙门氏菌、志贺氏菌、单核增生李斯特氏菌、致泻性大肠杆菌、变形杆菌等。目前,食品中致病菌的检测主要采用传统的细菌学培养方法,其操作繁琐,检测时间较长,通常需要4-7d才能完成,且灵敏度较低。近年来,PCR针对某一种或一类致病菌的检验方法被建立起来,但是一旦检测的方向有误就会造成时间和药品的浪费,急需建立一种快速有效且通量较高的检测方法。因此,多重PCR作为一种可同时检测多种病原菌的方法发展十分迅速。本研究建立了志贺氏菌、沙门氏菌、变形杆菌的多重PCR技术,根据志贺氏菌的侵染性质粒抗原H基因ipaH、沙门氏菌的侵染性质粒抗原B基因ipaB、变形杆菌的atpD基因设计了三对特异性引物,进行多重PCR反应,实现了对三种食源性致病菌的同时检测。试验过程中对多重PCR反应体系中引物添加量、镁离子添加量、dNTPmixture添加量及退火温度和Taq酶的添加量等影响要素进行优化,确定了适宜的多重PCR反应体系和扩增程序,其反应体系为:10×Buffer 2.5μL,50mM Mg2+ 1μL,2.5 mM dNTP 1μL,10μM的上下游引物各1μL,5 U/μL Taq酶0.3μL,双蒸水补足至25μL;多重PCR扩增程序为:95℃预变性5 min,94℃变性45 s,56℃退火45 s,72℃延伸45 s,30个循环,72℃终延伸10 min,反应产物在1.5%的琼脂糖凝胶中进行电泳,凝胶成像系统观察结果。对多重PCR扩增产物进行了克隆和DNA测序,登录Genebank将测序结果进行网上blast,从而证实PCR扩增产物确为目的扩增产物。本研究共检测了15株菌,2株志贺氏菌、4株沙门氏菌、2株变形杆菌均为阳性结果;7株其它肠杆菌均为阴性结果,从而验证多重PCR引物特异性。将三种致病菌混合,分别加入1对,2对和3对引物进行扩增,均能够准确检测出结果,因此该多重PCR反应特异性较强。本方法对三种食源性致病菌的纯菌培养物单菌检测灵敏度为志贺氏菌102CFU/ml、沙门氏菌101 CFU/ml、变形杆菌102 CFU/ml;同时检测三种食源性致病菌的灵敏度为志贺氏菌102 CFU/ml、沙门氏菌102 CFU/ml、变形杆菌102 CFU/ml。本方法使用试剂盒处理样品,再通过多重PCR方法同时检测人工污染猪肉中三种致病菌,志贺氏菌检出限为102 CFU/ml、沙门氏菌检出限为103 CFU/ml、变形杆菌检出限102 CFU/ml。并且检测可以在6 h内完成,大大缩短了检测时间。为多重PCR快速检测食品中三种食源性致病菌构建了一个技术平台。更多还原

【Abstract】 Food safety emergencies are high frequency in recent years.In these problems,the food poisoning result of Bacterial Pathogens was the most harmful.According to data came from hospitals and inspection-quarantine departments.Rapid detection of food-borne bacteria is essential to efficiently prevent pathogens prevalence and food poisoning.The main bacterial pathogens were Staphylococcus aureus,Salmonella spp., Shigella spp.,listeria monocytogenes, Proteus vulgaris and so on. Since contamination levels are generally low in foods,detection methods require lengthy culture enrichment steps to increase target bacterial numbers before isolation and identification using standard cultural procedues.These conventional food microbiological techniques often require several days to detect bacterial pathogens present in foods at low levels.With the advent of PCR,Individual PCR assays have been developed for detection and identification of the bacterial pathogens.but a large number of individual PCR assays would be necessary if single primer sets are used in separate reactions on a large number of food samples,which can be a relatively costly and time- consuming process.To reduce the number of tests needed for diagnosis of Individual PCR assays,the simultaneous detection of several pathogens with a multiplex PCR (m-PCR) approach would be relatively rapid and cost-effective.So multiplex PCR which can simultaneously detect more than one bacterium has been applied increasingly conmprehensive.In this study,m-PCR assays were developed for the simultaneous detection of Shigella spp., Salmonella spp. and Proteus vulgaris,took use of Shigell .spp ipaH gene, Salmonella spp ipaB gene,and atpD gene of Proteus vulgaris,according to the result of BLAST to design three special primers.This m- PCR assay was developed for the simultaneous detection of Shigella spp,Salmonella spp and Proteus vulgaris from meat.In the PCR process,DNA concentration,Mg2+ concentration,template volume,annealing temperature and Taq enzyme concentration are optimized to determine the optimal PCR.The reaction mixture consisted of 25μL: 2.5μL10×PCR buffer,50 mMMg2+ 1μL,1μL mixture of dNTPs,1μL of 10μM forward primer(each),1μL of 10μM reverse primer(each),0.3μL (5 U/μL)Taq enzyme,ddH2O 11.5μL.The reaction was run under the following conditions:Cool start;DNA pre-denaturation at 95℃for 5min , DNA denaturation at 94℃for 45 s,primer annealing at 56℃for 45 s,and DNA extention at 72℃for 45 s,run 30 cycles;the final extention was performed at 72℃for 10min.The PCR products were examined by l.5% agarose gel electrophoresis.PCR products were confirmed by DNA sequencing.The result of sequencing compared with the target gene sequence at gene bank showed that PCR amplified products were certified.There were 15 bacterial strains to be detected including 2 Shigella spp,4 Salmonella spp,1 Proteus vulgaris,and 7 other bacterial strains in order to determine specificity of amplification of primers.The results of 2 Shigella spp, 4 Salmonella spp and 1 Proteus vulgaris were positive and those of 7 other strains were negative.The specificity of the m-PCR assay was evaluated by testing the three primer sets with the purified DNAs of all the strains(separately or in different combinations) indicated above.Positive PCR amplification of DNA templates from Shigellas app,Salmonella spp,and Proteus vulgaris produced a single fragment,of the expected,for each pathogen.The three bacterial pathogens were simultaneously amplified.The sensitivity of m-PCR for the detoction of one of three bacterial pathogens when using purified DNA of bacterial type strains was 102 CFU/ml for Shigella spp, 101 CFU/ml for Salmonella spp,and 102 CFU/ml for Proteus vulgaris.The sensitivity of the simultaneous detection of the three bacterial pathogens with m-PCR when using purified DNA of the bacterial type strains was 102 CFU/ml for Shigella spp, 102 CFU/ml for Salmonella spp,and 102 CFU/ml for Proteus vulgaris.The bacterial pathogens DNA was directly extracted using DNA KIT.The extracted DNA was suitable for m-PCR detection.The detection limit of the m- PCR assay for meat was102 CFU/ml for Shigella spp, 103 CFU/ml for Salmonella spp,and 102 CFU/ml for Proteus vulgaris.The whole expe rimental procedure can be completed only 6 hours,shorter than the traditional biochemistry detection.The developed method in this assay allows detection of the pathogens in meat in less than 6h.This method is useful for the detection of Listeria monocytogenes infood.更多还原