用生物素标记核酸探针检测鼠金黄色葡萄球菌的研究

Detection of Staphylococcus aureus in Laboratory Animals by Dot Blotting Using Biotin-labeled nuc Gene Fragment as the Probe

利用聚合酶链反应技术从金黄色葡萄球菌基因组DNA中扩增出 6 78bp耐热核酸酶nuc基因特异性片段 ,经生物素标记后作为核酸斑点杂交试验的探针 ,对实验动物的金黄色葡萄球菌及其临床样品进行了检测。结果显示 ,标记探针与金黄色葡萄球菌DNA呈杂交阳性 ,而与链球菌、大肠埃希氏菌和巴氏杆菌DNA呈杂交阴性 ,斑点杂交的检测灵敏度为 1pg基因组DNA。用斑点杂交试验和PCR对 2 4 0份临床样品进行了检测 ,检出率为 2 9% (7 2 4 0 ) ,符合率为 10 0 %。这些试验结果表明 ,研究制备的核酸探针及建立的斑点杂交试验具有较高的特异性和敏感性 。

To establish a sensitive dot blotting for detecting Staphylococcus aureus(S.aureus), a DNA fragment of 678 bps of the nuc gene was amplified from S.aureus genome DNA by Polymerase Chain Reaction(PCR) and labeled with biotin as the probe.The purified genomic DNAs of S.aureus,Streptococcus,Escherichia coli and Pasteurella were blotted onto hybridization membrane and hybridized under high stringency conditions.The Biotin\|labeled probe reacted positively with the S.aureus genomic DNAs,but not with that of the non\| S.aureus with a sensitivity of 1 pg.240 clinical samples taken randomly from laboratory mice and rats were submitted to detection by the dot blotting and previously established PCR with a positive detection rate of 2.9%(7/240) and an agreement of 100%.These data demonstrated that the established dot blotting was a quick,sensitive and specific method for detection of S.aureus in laboratory animals.