摘要
目的 建立核酸探针检测实验动物金黄色葡萄球菌的方法。方法 将重组质粒pGEM NUC中金黄色葡萄球菌nuc基因特异性片段酶切回收 ,经生物素标记后作为核酸斑点杂交试验探针 ,对细菌菌株及临床样品进行了检测。结果 核酸探针与金黄色葡萄球菌DNA呈杂交阳性 ,而与表皮葡萄球菌、化脓性链球菌、大肠埃希菌等其他细菌DNA呈杂交阴性 ,斑点杂交检测的灵敏度为 1pg基因组DNA。用斑点杂交试验和PCR对 32 2份SPF小鼠的临床样品进行了检测 ,检出率为 3 1% (10 /32 2 ) ,符合率为 10 0 % ,与细菌学检查的结果相一致。结论 建立的DNA探针检测金黄色葡萄球菌方法具有高度的特异性和敏感性 ,且较快速 ,可用于实验动物金黄色葡萄球菌的监测。
Objective To establish a sensitive and specific molecular biological method for detecting Staphylococcus aureus (S. aureus, SA) in laboratory animals. Methods A DNA fragment of 676 bps of the nuc gene was applified from SA genome DNA by PCR and labeled with biotin as the probe. The purified genomic DNAs of SA1800, SAm0109 and Staphylococcus epidermidis, Streptococcus pyogenes, Escherichia coli were blotted onto the Hybond N++ nylon membrane, fixed using UV irradiation, hybridized under high stringency conditions, and detected using the Enhanced Chemiluminesence kit (ECL). Results The probe reacted positively with the SA genomic DNAs, but not with that of the non-SA with a sensitivity of I pg. 322 clinical samples taken randomly from specific pathogen free (SPF) mice were submitted to detection by the dot blotting and previously established PCR and culture with a positive detection rate of 3.1% (10/322) and an agreement of 100 %. Conclusion The dot-hybridization assay was a new rapid, sensitive and specific method and could be applied to detect SA in laboratory animals.
出处
《中国比较医学杂志》
CAS
2004年第5期301-303,共3页
Chinese Journal of Comparative Medicine
基金
国家"十五"科技攻关计划"国家遗传工程小鼠资源库的建立"(No .2 0 0 1BA70 113 )
