摘要
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，设计3对特异性引物进行多重PCR并对反应条件进行优化。结果表明3对引物能特异地扩增出284bp、622bp、484bp的目的条带；最佳反应条件为沙门菌、大肠杆菌、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2001μmol／L，Taq DNA聚合酶1.5u，退火温度55．0℃-57．4℃之间；在此条件下多重PCR同时检测DNA的敏感性分别是10．2pg、10．2pg、102．0pg，检测时间4h。建立的多重PCR是一种敏感、特异、准确、快速的方法，为同时检测食品中沙门菌、大肠杆菌和金黄色葡萄球菌奠定了基础。
According to DNA sequences of the invA gene of Salmonella spp. , the phoA gene of Escherichia coli and the nuc gene of Staphylococcus aureus, three pairs; of aligonucleotide primers were designed and synthesized to amplify the special DNA sequences by multiplex PCR. Moreover, the reaction conditions of multiplex PCR were optimized. The results showed the multiplex PCR. using the three pairs of primers produced specific amplicons of expected sizes, 284hp for Salmonella spp. , 622bp for Escherichia coli, 484bp for Staphylococcas aureran. The optimized reaction conditions followed as the concentration of primer 40nmnl/L for Salmonella spp. , 40nmol/L for Escherichia coli, 80nmol/L for Staphyiococcus aureus, 2.4 mmol/L Mg^2＋ , 2001μmol/L dNTP, 1.5U Taq DNA polymerase, anneal temperature from 55.0℃ to 57.4℃. Under the condition, the detection limits fur DNA template were 10.2pg, 10. 2pg and 102. 0pg for Salmonella spp. , Escherichia coli and Staphylococcus aureas, respectively. The whole process could be completed within 4h. The multiplex PCR assay was a specific, sensitive, rapid and reliable method for detecting Salmonella spp. , Escherichia cull and Staphylococcus aureus, which establish imnortant foundation for simultaneous detection for these three bacteria in food.
出处
《微生物学通报》
CAS
CSCD
北大核心
2006年第6期89-94,共6页
Microbiology China
基金
湖北省科技厅资助项目(No.2004AA203B03)
关键词
多重PCR
沙门菌
大肠杆菌
金黄色葡萄球菌
Multiplex PCR, Salmonella spp. , Escherichia coli, Staphylococcus aureus
