金黄色葡萄球菌中毒性休克综合征毒素-1 PCR检测方法的建立与初步应用  

Establishment and preliminary application of a PCR method for detection of toxic shock syndrome toxin-1 in Staphylococcus aureus

作　　者：张晓丽 陈卓 于佳鑫 孙悦青 王洋 栾剑[1] 周晓梅[1] 郭海勇[1] ZHANG Xiaoli;CHEN Zhuo;YU Jiaxin;SUN Yueqing;WANG Yang;LUAN Jian;ZHOU Xiaomei;GUO Haiyong(School of Life Science,Jilin Normal University,Siping 136000,China)

机构地区：[1]吉林师范大学生命科学学院

出　　处：《河北农业大学学报》2019年第6期97-102,共6页Journal of Hebei Agricultural University

基　　金：吉林省教育厅“十三五”科学技术研究规划项目(JJKH20170382KJ);吉林师范大学大学生科研基金项目(17184)

摘　　要：试验旨在建立敏感、特异快速检测金黄色葡萄球菌超抗原中毒性休克综合征毒素-1(TSST-1)的PCR方法。根据TSST-1毒素基因(tst基因)的序列,设计合成了特异性引物,优化了PCR方法的引物浓度、退火温度及DNA模板量,测定了方法的灵敏度与特异性,并应用所建立的PCR方法检测了67株金黄色葡萄球菌分离株中的tst基因。结果表明,建立的PCR方法的最佳退火温度为55℃、引物浓度为0.4μmol/L,检测灵敏度为20 pg DNA,与表皮葡萄球菌、猪链球菌、四联球菌、沙门氏菌和大肠杆菌等均不发生交叉反应。在67株金黄色葡萄球菌分离株中,13株携带tst基因,其中鸡源分离株中携带tst基因的菌株约占9.1%(1/11),牛源分离株中携带tst基因的菌株约占21.4%(12/56)。本研究建立的PCR方法能够快速、特异、敏感地检测金黄色葡萄球菌超抗原TSST-1毒素基因,为动物源性食品中TSST-1的检测及产TSST-1毒素菌株的流行病学调查提供重要技术支持。This study was aimed to establish a sensitive,specific and rapid PCR method for the detection of superantigenic toxic shock syndrome toxin-1(TSST-1)in Staphylococcus aureus.A pair of specific primer was designed and synthesized according to the reported sequence of TSST-1 gene(tst gene).A PCR method for tst gene detection was established by optimizing concentration of primers,annealing temperature and concentration of template.The sensitivity and specificity of the method were tested.Then,the PCR method was used to detect tst gene in 67 clinical isolates of S.aureus.The test results indicated that the optimized annealing temperature of the developed PCR method was 55℃,and the concentration of primers was 0.4μmol/L.The PCR method had a detection limit of 20 pg for DNA of S.aureus.There was no cross reaction with Staphylococcus epidermidis,Streptococcus suis,Mlicrococcus tetragenus,Salmonella and Escherichia coli.13 out of 67 isolates harbored tst gene.The percentage of tst gene in chicken-original isolates was about 9.1%(1/11)and 21.4%(12/56)in bovine derived isolates.The developed method with good sensitivity and specificity can be used to detect superantigenic tst gene in S.aureus,which would provide an important technical support for detecting the TSST-1 in animal derived foods and the epidemiological investigation of Staphylococcus aureus isolated with tst gene coding TSST-1.

关 键 词：金黄色葡萄球菌 超抗原 中毒性休克综合征毒素-1 PCR 

分 类 号：S852.61[农业科学—基础兽医学]