金黄色葡萄球菌肠毒素快速分型检测的研究  

Study on rapid detection of Staphylococcal enterotoxin

作　　者：魏泉德[1] 黄辉涛 林毅雄 李红霞[1] 

机构地区：[1]珠海市疾病预防控制中心,广东珠海519000

出　　处：《热带医学杂志》2015年第9期1191-1195,共5页Journal of Tropical Medicine

基　　金：珠海市科技计划项目(2013D0401990024)

摘　　要：目的建立能在同一PCR反应条件下同时检测葡萄球菌肠毒素(SEs)5个亚型(SEA、SEB、SEC、SED、SEE)的PCR快速检测方法,用于金黄色葡萄球菌引起的食源性疾病的快速诊断。方法根据已公布的SEA、SEB、SEC、SED、SEE的基因序列,利用Vector NTI suite 8软件设计SEABCE的通用PCR引物以及针对SED的特异性引物进行多重PCR扩增;再对SEA、SEB、SEC、SEE进行扩增,通过优化反应条件,建立在同一PCR反应条件下同时检测SEA^E肠毒素的PCR检测方法 ,同时进行该方法的特异性及灵敏度分析,扩增产物进行测序鉴定。结果 SEABCE、SED多重PCR扩增产物均有预期大小的目的 DNA片段,分别为157、340 bp;SEA、SEB、SEC、SEE的PCR分型扩增显示均有预期大小的目的 DNA片段,分别为579、602、601、125 bp;SEA、SEB、SEC、SED、SEE基因寡核苷酸引物的特异性分析均出现预期大小的目的片段,对照菌未出现;测序结果表明与相应的靶基因序列同源性达100%。本方法灵敏度达0.8~1.0×102 CFU/ml,与其他PCR单个分型检测肠毒素的敏感性相仿,特异性较好,能很好的确定金黄色葡萄球菌肠毒素引起的食物中毒。结论本检测方法简便、快捷,可用于金黄色葡萄球菌肠毒素食物中毒的快速诊断和肠毒素的分型检测。Objective To establish a method of rapid detection of SEA,SEB,SEC,SED,and SEE under the same PCR reaction, which can be applied to the rapid diagnosis of foodborne diseases caused by Staphylococcus aureus. Methods According to the gene sequences of SEA, SEB, SEC, SED, and SEE, common PCR primers for SEA, SEB, SEC and SEE,and SED specific PCR primers were designedwith the help of the software Vector NTI suite 8.SEA, SEB, SEC,SED and SEE were emplified in the same PCR reaction conditions. By optimizing the reaction conditions, we established PCR for detection of SEA-E.Meanwhile, on the specificity and sensitivity of the assay was analyzed and the amplified products was confirmed by sequencing identification. Results Target DNA fragments appear in the SEABCE,SED multiple PCR amplification products, respectively 157 and 340 bp. Target DNA fragments appear in the typing amplification of SEA,SEB,SEC and SEE, respectively 579,602,601,125 bp; Target DNA fragments appear in the specificity of the assay,but not in the control bacteria.The sequencing results show that the homology of target gene is100%. This method had a good specificity and can identify food poisoning caused by Staphylococcal enterotoxin. The sensitivity reached 0.8 ×1012~1.0 ×102CFU / m L which was similar to other reports of PCR method. Conclusion This novel method can be applied to the rapid diagnosis of food poisoning caused by Staphylococcal enterotoxin and the subtype enterotoxin.

关 键 词：PCR 金黄色葡萄球菌 肠毒素 

分 类 号：R378.11[医药卫生—病原生物学]