PCR技术在检测鼠金黄色葡萄球菌中的应用研究

Study on the Application of Polymerase Chain Reaction for Detecting of Staphylococcus aureus in Laboratory Rats and Mice

目的 建立实验大小鼠金黄色葡萄球菌的快速检测方法———PCR法。方法 根据已公布的金黄色葡萄球菌耐热核酸酶nuc基因的序列 ,设计并合成一对特异性的引物 ,利用PCR技术扩增nuc基因片段。对金黄色葡萄球菌和其他非金黄色葡萄球菌菌株抽提的DNA进行扩增。结果 金黄色葡萄球菌PCR产物出现 6 6 8bp的特异性DNA扩增片段 ,而其他非金黄色葡萄球菌未出现扩增片段 ,证实了合成的引物对金黄色葡萄球菌具有特异性。将抽提的金黄色葡萄球菌DNA进行系列稀释 ,测定此PCR体系的敏感性 ,结果显示 ,该PCR体系能检出 3pg金黄色葡萄球菌DNA ,且从抽提DNA到PCR扩增及电泳结束仅需 4h。结论 本研究所建立的扩增耐热核酸酶nuc基因检测鼠金黄色葡萄球菌的PCR方法 ,具有快速、可靠、敏感和特异的特点 ,可用于临床样品和金黄色葡萄球菌感染时的检测 ,适合应用于实验大小鼠的监测。

Objective To establish a rapid method for detection of Staphylococcus aureus in laboratory rats and mice ——Polymerase Chain Reaction (PCR). Methods According to the sequence of the nuc gene, which encodes the thermostable nuclease of Staphylococcus aureus, a pair of oligonucleotide primers special to the sequence of nuc gene were designed and synthesized, and were used to amplify a sequence of nuc gene by PCR. DNA extracted from Staphylococcus aureus was amplified and DNA from non-Staphylococcus aureus was used for control. Results The PCR product was detected by agarose gel electrophoresis analysis. The Staphylococcus aureus revealed positive results of the presence of a 668 bp specific fragment on gel, while non-Staphylococcus aureus gave negative results demonstrating that the primers should be specific for Staphylococcus aureus tested. The PCR would be able to detect as little as 3 pg Staphylococcus aureus DNA as determined by amplification of serially diluted Staphylococcus aureus DNA. The whole process of DNA extraction, amplification and electrophoresis could be finished within 4 hours. Conclusion The results showed the developed PCR assay was a rapid, reliable, sensitive and specific method of detecting Staphylococcus aureus infections in rats and mice.