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目的建立能在同一PCR反应条件下同时检测SEA、SEB、SEC、SEE的PCR快速检测方法,以用于金黄色葡萄球菌引起的食源性疾病的快速诊断。方法根据Gen Bank公布的SEA、SEB、SEC、SEE基因的保守序列,利用Vector NTI suite 8软件设计SEA/SEE、SEB/SEC通用PCR引物来特异性扩增相应的基因片段,通过优化反应条件,建立在同一PCR反应条件下同时检测SEs各型的PCR检测方法,同时进行常见食源性致病菌的特异性分析及灵敏度分析,扩增产物进行测序鉴定。结果金黄色葡萄球菌肠毒素SEA、SEE引物扩增后电泳片段位于目的片段330 bp处,SEB、SEC片段位于目的片段258 bp处,SEA、SEB、SEC和SEE基因PCR产物的电泳片段位于目的片段的579、602、601和125 bp处,结果均显示引物扩增和PCR扩增产物均有预期大小的目的 DNA片段。本方法中的基因引物的PCR扩增金黄色葡萄球菌、肠炎沙门菌、鼠伤寒沙门菌、伤寒沙门菌、痢疾志贺菌、大肠杆菌O157:H 7、副溶血性弧菌、奇异变形杆菌、福氏志贺菌、肠毒素型大肠埃希菌、肠侵袭型大肠埃希菌、肠致病型大肠埃希菌、肠出血型大肠埃希菌均未出现预期大小的目的条带。本方法敏感性达80~100 CFU/m L。结论本检测方法所建立的PCR可用于金黄色葡萄球菌肠毒素A、B、C、E型别食物中毒的快速诊断和分型检测。
Objective To establish a rapid PCR method for the simultaneous detection of SEA, SEB, SEC and SEE under the same PCR reaction conditions for the rapid diagnosis of foodborne diseases caused by Staphylococcus aureus. Methods According to the conserved sequences of SEA, SEB, SEC and SEE genes published by Gen Bank, vector NTI suite 8 software was used to design SEA / SEE and SEB / SEC universal PCR primers to amplify the corresponding gene fragments. By optimizing the reaction conditions, The detection method of simultaneous detection of various types of SEs under the same PCR reaction conditions was established. At the same time, the specificity analysis and sensitivity analysis of common food-borne pathogens were carried out. The amplified products were identified by sequencing. Results The amplified fragment of SEA and SEE was located at 330 bp. The SEB and SEC fragments were located at 258 bp. The electrophoresis fragments of SEA, SEB, SEC and SEE genes were located in the target fragment 579,602,601 and 125 bp, the results showed that the primer amplification and PCR amplification products have the desired size of the DNA fragment. The gene primers of this method amplify S. aureus, S. Enteritidis, S. typhimurium, S. typhi, S. dysenteriae, E. coli O157: H7, V. parahaemolyticus, Shigella flexneri, enterotoxigenic Escherichia coli, intestinal invasive Escherichia coli, enteric pathogenic Escherichia coli, enterohemorrhagic Escherichia coli did not appear the desired size of the target band. The method sensitivity of 80 ~ 100 CFU / m L. Conclusion The PCR established by this test method can be used for the rapid diagnosis and typing detection of Staphylococcus aureus enterotoxins type A, B, C and E food poisoning.