作　　者： ; ; ; ; ; ; ; (吴永生）; (何浩）; (罗公平）;

机构地区： 绥芬河进出口检验检疫局黑龙江绥芬河

出　　处： 《中国预防兽医学报》 2006年第4期436-440,445,共6页

摘　　要： 本研究根据金黄色葡萄球菌(Staphylococcusaureus,SA)耐热核酸酶(nuc)基因、伤寒沙门氏菌(Salmonellatyphi,ST)鞭毛抗原(H1-d)基因和副溶血性弧菌(Vibrioparahaemolyticus,VP)热稳定直接溶血素(tdh)基因,分别设计了三对引物Nuc-F/Nu-R、H1-d-F/H1-d-R和Tdh-F/Tdh-R,预计PCR扩增的DNA片断分别为279bp、202bp和458bp。通过对单个基因PCR和单管多重PCR扩增的特异性、敏感性分析以及对单管多重PCR扩增条件如引物浓度、退火温度和dNTP浓度等的优化,建立了快速检测金黄色葡萄球菌、伤寒沙门氏菌和副溶血弧菌的单管多重PCR方法,该方法检测的敏感性分别为:47.8pgST的基因组DNA,22.7pgSA的基因组DNA和0.3745pgVP的基因组DNA。模拟试验显示最低检测限度为分别为:SA150CFU/反应体系,ST98CFU/反应体系,VP53CFU/反应体系。表明该方法具有很好的应用价值和开发前景。 Three pairs of primers have been designed according to the Staphylococcus aureus （SA） nuc gene, Salmonella typhi （ST） Hl-d gene and Vibrio parahaemolyticus （VP） tdh gene, designated Nuc-F/Nu-R, HI-d-F/HI-d-R and Tdh-F/Tdh-R, respectively. Anticipated PCR product is 279 bp, 202 bp and 458 bp. Multiple PCR method has been development for diagnose Staphylococcus aureus, Salmonella typhi and Vibdo parahaemolyticus, after analysis and optimization reaction condition, e.g., the specificity and sensitivity of PCR for single gene and multiple PCR in a single tube, concentration of primers and dNTP, anneal temperature. The sensitivity of multiple PCR is 47.8 pg genome DNA for ST, 22.7 pg for SA and 0.3745 pg for VP. Simulation experiment showed that the minimum detection limit is 150 CFU for SA, 98 CFU for ST and 53 CFU for VP. It indicates that the multiple PCR method is valuable for exploitation and application.

关 键 词： 多重 金黄色葡萄球菌 副溶血性弧茵 伤寒沙门氏菌

领　　域： [农业科学] [农业科学] [农业科学]