多重PCR检测沙门菌、大肠杆菌和金黄色葡萄球菌的研究
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摘要
食品中污染的病原细菌是引起食源性疾病的主要因素之一，快速检测食品中病原细菌是及时有效预防病原细菌传播及食物中毒的重要前提。目前病原细菌的检测主要依靠常规的细菌学培养方法，一般需4～7d，操作繁琐，费时耗力：多重PCR作为一种可同时检测多种病原细菌的方法发展十分迅速。本研究建立了沙门菌、大肠杆菌和金黄色葡萄球菌的多重PCR检测技术，并初步应用于湖北省出入境检验检疫局部分出口食品样品的检测中，主要研究结果如下：
1 沙门菌、大肠杆菌和金黄色葡萄球菌多重PCR引物设计及特异性分析(1)根据沙门菌invA基因、大肠杆菌phoA基因和金黄色葡萄球菌nuc基因序列，应用Primer 5.0设计引物并通过Oligo 6.0进行分析，确保引物之间无二聚体形成，并对各引物间的互补性、最佳退火温度进行了分析，同时应用BLAST程序，通过GeneBank对各引物及扩增产物进行同源性对比，获得三对特异性引物。(2)优化沙门菌、大肠杆菌和金黄色葡萄球菌PCR反应条件，进行了引物特异性实验，结果设计的沙门菌、大肠杆菌和金黄色葡萄球菌引物能特异的扩增出284bp、622bp、484bp的目的条带，对照菌株均为阴性。引物间无交叉反应，特异性好。
2 沙门菌、大肠杆菌和金黄色葡萄球菌多重PCR检测方法的建立
经过优化实验确定了多重PCR最佳反应条件为沙门菌、大肠杆菌和金黄色葡萄球菌的引物浓度分别为40nmol／L、40nmol／L、80nmol／L，Mg~(2+)浓度2.4mmol／L，dNTP浓度200μmol／L，Taq DNA聚合酶1.5U。循环参数为：94℃预变性7min；94℃变性30s，55℃退火30s，72℃延伸30s，共30个循环；最后72℃延伸5min。在此条件下多重PCR同时检测沙门菌、大肠杆菌和金黄色葡萄球菌DNA的敏感性分别是10.2pg、10.2pg、102pg。
3 同时富集沙门菌、大肠杆菌和金黄色葡萄球菌增菌培养基的研究
本研究设计出一种能同时富集培养沙门菌、大肠杆菌和金黄色葡萄球菌的缓冲盐水肉汤(buffed saline broth，BSB)，其主要组分和含量为：蛋白胨10g、牛肉膏3g、磷酸氢二钠(Na_2HPO_4·12H_2O)9g、磷酸二氢钾1.5g、添加剂50g、蒸馏水1000mL，pH7.2。
4 多重PCR检测方法的应用与评价
对10份人为接种病原菌牛奶样品、20份污水样品和50份湖北省出入境检验检疫局出口虾样品，多重PCR和国标方法均有9份沙门菌阳性，27份大肠杆菌阳性，10份金黄色葡萄球菌阳性，多重PCR阳性而国标方法阴性的有1份沙门菌、3份大
Food contamination with bacterial pathogens are major reasons of food-borne disease. Rapid detection of food-borne bacteria is essential to efficiently prevent pathogens prevalence and food poisoning. Conventional methods of detecting bacteria in food relies on selective medium, microbiological and biochemical test can often take more than 4～7 days to complete, which are cumbersome and time-consuming. Multiplex PCR which can simultaneously detect more than one bacterium has been applied increasingly comprehensive. In this study, multiplex PCR assays were developed for the simultaneous detection of Salmonella spp., Escherichia coli and Staphylococcus aureus and used in some food samples from HuBei entry-exit inspection and quarantine bureau. The results are as follows:
1. Results of primer design for multiplex PCR and specificity test
(1) According to the invA gene of Salmonella spp., the phoA gene of Escherichia coli and the nuc gene of Staphylococcus aureus, three pairs of primers were designed by Primer 5.0 and were analyzed by Oligo 6.0 to insure the close anneal temperature and avoid the formation of steady dimer. Moreover the primers and amplification fragments were analyzed by BLAST to avoid high homology and complementarity through GeneBank. (2)The PCR amplification conditions were optimized and the primers specificity test were developed. The results showed that the three pairs of primers produced specific amplicons of expected sizes, 284bp for Salmonella spp., 622bp for Escherichia coli, 484bp for Staphylococcus aureus.
2. Development of multiplex PCR assay for Salmonella spp., Escherichia coli and Staphylococcus aureus
The optimized reaction conditions followed as the concentration of primer 40nmol/L for Salmonella spp., 40nmol/L for Escherichia coli, 80nmol/L for Staphylococcus aureus, 2.4mmol/L Mg~(2+), 200 μ mol/L dNTP, 1.5U Taq DNA polymerase. The reaction was run under the following conditions: DNA pre-denaturation at 94 °C for 7min, DNA denaturation at 94 °C for 30s, primer annealing at 55 °C for 30s, and DNA extension at 72 °C for 30s for 30 cycles, the last extension was performed at 72°C for 5min. Under the
1 沙门菌、大肠杆菌和金黄色葡萄球菌多重PCR引物设计及特异性分析(1)根据沙门菌invA基因、大肠杆菌phoA基因和金黄色葡萄球菌nuc基因序列，应用Primer 5.0设计引物并通过Oligo 6.0进行分析，确保引物之间无二聚体形成，并对各引物间的互补性、最佳退火温度进行了分析，同时应用BLAST程序，通过GeneBank对各引物及扩增产物进行同源性对比，获得三对特异性引物。(2)优化沙门菌、大肠杆菌和金黄色葡萄球菌PCR反应条件，进行了引物特异性实验，结果设计的沙门菌、大肠杆菌和金黄色葡萄球菌引物能特异的扩增出284bp、622bp、484bp的目的条带，对照菌株均为阴性。引物间无交叉反应，特异性好。
2 沙门菌、大肠杆菌和金黄色葡萄球菌多重PCR检测方法的建立
经过优化实验确定了多重PCR最佳反应条件为沙门菌、大肠杆菌和金黄色葡萄球菌的引物浓度分别为40nmol／L、40nmol／L、80nmol／L，Mg~(2+)浓度2.4mmol／L，dNTP浓度200μmol／L，Taq DNA聚合酶1.5U。循环参数为：94℃预变性7min；94℃变性30s，55℃退火30s，72℃延伸30s，共30个循环；最后72℃延伸5min。在此条件下多重PCR同时检测沙门菌、大肠杆菌和金黄色葡萄球菌DNA的敏感性分别是10.2pg、10.2pg、102pg。
3 同时富集沙门菌、大肠杆菌和金黄色葡萄球菌增菌培养基的研究
本研究设计出一种能同时富集培养沙门菌、大肠杆菌和金黄色葡萄球菌的缓冲盐水肉汤(buffed saline broth，BSB)，其主要组分和含量为：蛋白胨10g、牛肉膏3g、磷酸氢二钠(Na_2HPO_4·12H_2O)9g、磷酸二氢钾1.5g、添加剂50g、蒸馏水1000mL，pH7.2。
4 多重PCR检测方法的应用与评价
对10份人为接种病原菌牛奶样品、20份污水样品和50份湖北省出入境检验检疫局出口虾样品，多重PCR和国标方法均有9份沙门菌阳性，27份大肠杆菌阳性，10份金黄色葡萄球菌阳性，多重PCR阳性而国标方法阴性的有1份沙门菌、3份大
Food contamination with bacterial pathogens are major reasons of food-borne disease. Rapid detection of food-borne bacteria is essential to efficiently prevent pathogens prevalence and food poisoning. Conventional methods of detecting bacteria in food relies on selective medium, microbiological and biochemical test can often take more than 4～7 days to complete, which are cumbersome and time-consuming. Multiplex PCR which can simultaneously detect more than one bacterium has been applied increasingly comprehensive. In this study, multiplex PCR assays were developed for the simultaneous detection of Salmonella spp., Escherichia coli and Staphylococcus aureus and used in some food samples from HuBei entry-exit inspection and quarantine bureau. The results are as follows:
1. Results of primer design for multiplex PCR and specificity test
(1) According to the invA gene of Salmonella spp., the phoA gene of Escherichia coli and the nuc gene of Staphylococcus aureus, three pairs of primers were designed by Primer 5.0 and were analyzed by Oligo 6.0 to insure the close anneal temperature and avoid the formation of steady dimer. Moreover the primers and amplification fragments were analyzed by BLAST to avoid high homology and complementarity through GeneBank. (2)The PCR amplification conditions were optimized and the primers specificity test were developed. The results showed that the three pairs of primers produced specific amplicons of expected sizes, 284bp for Salmonella spp., 622bp for Escherichia coli, 484bp for Staphylococcus aureus.
2. Development of multiplex PCR assay for Salmonella spp., Escherichia coli and Staphylococcus aureus
The optimized reaction conditions followed as the concentration of primer 40nmol/L for Salmonella spp., 40nmol/L for Escherichia coli, 80nmol/L for Staphylococcus aureus, 2.4mmol/L Mg~(2+), 200 μ mol/L dNTP, 1.5U Taq DNA polymerase. The reaction was run under the following conditions: DNA pre-denaturation at 94 °C for 7min, DNA denaturation at 94 °C for 30s, primer annealing at 55 °C for 30s, and DNA extension at 72 °C for 30s for 30 cycles, the last extension was performed at 72°C for 5min. Under the
引文
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