多重PCR快速检测金黄色葡萄球菌四型肠毒素基因的研究  

A STUDY ON FAST DETECTION OF FOUR KINDS OF STAPHYLOCOCCUS AUREUS ENTEROTOXINS GENES WITH A MULTIPLEX PCR METHOD

作　　者：杨玉军[1] 黄韦唯[2] 王志云[2] 刘衡川[2] 余倩[2] 汪川[2] 

机构地区：[1]攀枝花学院医学院基础医学教研室,攀枝花617000 [2]四川大学华西公共卫生学院医检教研室

出　　处：《现代预防医学》2009年第9期1713-1715,共3页Modern Preventive Medicine

摘　　要：[目的]建立一种快速、高效地检测金黄色葡萄球菌SEA、SEB、SEC和SED基因的多重PCR法并进行优化。[方法]试剂盒提取产肠毒素金黄色葡萄球菌基因组DNA作为模板,根据SEA、SEB、SEC和SED肠毒素基因片段保守区序列设计引物,多重PCR扩增产肠毒素金黄色葡萄球菌基因组中特异靶序列。对反应体系Mg2+浓度、引物浓度和退火温度进行优化,并初步检测方法特异性。[结果]在PCR反应体系中加入设计的4对引物可同时扩增出金黄色葡萄球菌四型肠毒素基因中的特异序列;在总体积为25μl的反应体系中,最优Mg2+浓度为2.0mmol/L,引物浓度分别为0.2μmol/L,退火温度为49℃;以大肠杆菌O157︰H7和肠炎沙门菌510041基因组DNA为模板扩增不出特异片段。[结论]成功建立金黄色葡萄球菌四型肠毒素基因多重PCR法,该法能快速、高效地同时检测SEA、SEB、SEC和SED基因,比传统PCR方法省时省力,可以弥补传统PCR法的不足。[Objective] To establish and optimize a multiplex PCR method for detecting SEA, SEB, SEC and SED genes of Staphylococcus aureus fast and highly effectively. [Methods] Genomic DNA of the enterotoxigenic S. aureus was ab-stracted with bacteria DNA abstract kit and used as the template for multiplex PCR. Primers were designed according to the conservative sequence of SEA, SEB, SEC and SED enterotoxin gene fragments and were used to amplify the target sequences in S. aureus genomic DNA. The Mg2＋ concentration, primers concentration in the reaction system and the annealing temperature were optimized, and the specificity of the method was examined. [Results] Using these four pairs of primers could amplify the specific sequences of four kinds of enterotoxin genes simultaneously; the optimum Mg2＋ concentration was 2.0mmol / L, primer con-centration was 0.2μmol / L respectively in the total volume of 25μl of the reaction system and optimum annealing temperature was 49℃; specific sequences could not be amplified when E. coli O157︰H7 and S. typhimurium genomic DNA were used as templates. [Conclusion] A multiple PCR method for detecting four kinds of S. aureus enterotoxin genes has been established. It could detect SEA, SEB, SEC and SED genes simultaneously, fast and highly effectively. Compared with traditional PCR methods, it seems more time-saving and labour-saving. It may make up for the shortcomings of traditional PCR methods.

关 键 词：多重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]