【作者】 陈珊珊；

【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， 2008， 硕士

【摘要】 阪崎肠杆菌能引起严重的新生儿脑膜炎、菌血症、小肠结肠炎,并可能引起神经功能紊乱,造成严重的后遗症,死亡率高达20%～50%以上,引起世界范围内的广泛关注。通过研究发现婴儿配方奶粉是其主要的感染源。传统方法操作繁琐,检测时间长,且灵敏度较低。本研究利用FTA滤膜与PCR技术相结合的方法建立了一套从婴儿配方奶粉中快速检出阪崎肠杆菌的方法,缩短了阪崎肠杆菌的检测时间,提高了灵敏度。本研究以阪崎肠杆菌的16S rRNA基因为靶基因,选择特异性引物,进行PCR扩增,检测婴儿配方奶粉中阪崎肠杆菌。对PCR反应体系中镁离子浓度、引物添加量及退火温度和循环数进行优化,以确定适宜的PCR反应体系和扩增程序,其适宜反应体系为:6μL10×PCRbuffer,4μLdNTPs混合物,2.5 Mg²⁺μL（25mM）,2μL10μmol正向引物,2μL10μmol反向引物,0.25μL（5U/μL）Taq,23.25μL水,总反应体系为50μL;其适宜的PCR扩增程序为:PCR反应采用冷启动,95℃预变性5 min;变性95℃1 min～退火61℃0.5 min进行35个循环。PCR反应产物在2%的琼脂糖凝胶中进行电泳,凝胶成像系统观察结果。对扩增产物进行了测序,PCR扩增产物序列与文献报道的靶基因序列的同源性达100%,从而证实PCR扩增产物确为目的扩增产物。本研究共检测了16株菌,以验证引物的特异性,结果表明:4株阪崎肠杆菌均为阳性结果;12株其它菌株均为阴性结果。因此该引物特异性强,可用于检测阪崎肠杆菌。采用FTA滤膜制备模板进行PCR扩增,其方法的灵敏度为7×10¹ cfu/mL。采用FTA滤膜制备模板,利用PCR技术检测婴儿配方奶粉中阪崎肠杆菌,以确定检出限。结果表明,经过4h增菌后婴儿配方奶粉中阪崎肠杆菌的检出限为7×10⁰cfu/100g。可在6h内完成对婴儿配方奶粉中阪崎肠杆菌的检测,比目前普遍采用的传统检测的方法缩短了12-22h。比较了检测婴儿配方奶粉中阪崎肠杆菌的6种模板制备方法,结果表明FTA滤膜法可高效从样品中提取阪崎肠杆菌DNA,可有效的消除PCR反应的抑制因子,灵敏度高、操作简便、耗时短,该方法明显优于其他方法。对实际样品进行检测,将本方法与行业标准SN/T 1632.1-2005方法进行比较,确定本方法敏感性为100%,特异性为98.2%符合率为98.2%。本研究方法使用FTA滤膜法制备模板DNA,可高效的提取样品中的DNA,有效的消除PCR反应抑制因子,操作简便,为PCR快速检测食品中的致病菌构建了一个技术平台。更多还原

【Abstract】 Enterobacter sakazakii is considered an opportunistic pathogen and has been implicated in food diseases causing meningitis、bacteremia or necrotizing enterocolitis. Specially in neonates and infants. The fatality rates have reached above 20%-50%.Only powered infant formula has been linked to the Enterobacter sakazakii and the sensitivity is lower. In this study, the use of PCR amplification with FTA filters was developed to detect Enterobacter sakazakii form infant formula quickly and the time of detection was shortened.In this study, took use of E. sakazakii of special sequences of conservative 16S rRNA to design a special primers. In the PCR process, Mg²⁺ concentration, annealing temperature and PCR circles are optimized to determine the optimal PCR. The reaction mixture consisted of 50μL: 6μL 10×PCR buffer, 4μL mixture of dNTPs, 2.5μL Mg²⁺ （25 mM）, 2μL of 10μmol forward primer, 2μL of 10μmol reverse primer, 0.25μL （5U /μL） Taq enzyme, ddH₂O 23.25μL. The reaction was run under the following conditions: Cool start, DNA pre-denaturation at 95℃for 5 min, DNA denaturation at 95℃for 1 min, primer annealing at 61℃, run 35 cycles. The PCR products were examined by 2% agarose gel electrophoresis. The result of sequencing compared with target gene sequence showed the homology got to 100%, so PCR amplified products were certified. There were 16 bacterial strains to be detected including four strains of E. sakazakii and 14 other bacterial strains except for E.sakazakii strain in order to determine specificity of amplification of primers. The results showed four strains belonging to positive results after primers extending, the results were negative for other strains. In this study result proved that the primers used in PCR was specific to detection E. sakazakii.The Sensitivity of PCR-based assay with FTA filters as templates was 7×10¹ cfu/mL.The Detection limit of E. sakazakii in reconstituted infant formula by using FTA filter for DNA template preparation after an enrichment step of 4 h was 7×10⁰ cfu/100g.The whole experimental procedure can be completed only 6 hours, shorter 12-22 h than the traditional biochemistry detection. The effect of six methods of extracting DNA from Enterobacter sakazakii in infant formula were compared, an efficient extraction procedure was confirmed for extraction of Enterobacter sakazakii DNA from infant formula. Enterobacter sakazakii DNA was extracted using FTA filter. The detection time of PCR method is significantly shorter than the other methods.In this study, real detection was made. The result indicates: the sensitivity of the method is 100%, the specificity is 98.2%, and the coincidence rate is 98.2%. Preparation of PCR templates with using FTA filters more sensitive and was able to efficiently remove inhibitors that could affect the PCR reaction. The results of this study demonstrate that a filter-based method can efficiently be used to detect food-borne bacterial pathogens in food.更多还原