摘要
目的利用PCR技术,无需增菌,直接检测乳品中的金黄色葡萄球菌。方法通过溶剂提取的方法从人工样品中提取模板,以金黄色葡萄球菌耐热核酸酶基因(nuc)为靶基因,经过PCR扩增得到279bp的产物。经过DNA测序证实该产物为目的扩增产物。采用PCR方法实际检测了乳品中的金黄色葡萄球菌,同时,与国标GB4789.10—94方法及两种快速检测致病性金黄色葡萄球菌测试片PetrifilmRSA.CountPlate及Baird-parker+RPFAgar进行了比较。结果PCR方法的灵敏度高,全脂乳和脱脂乳检测的检出限为10CFU/ml,奶酪检测的检出限为55CFU/g,可在6h内完成对乳品中金黄色葡萄球菌的检测,比目前普遍采用的先增菌再进行PCR检测的方法缩短了12～24h。与国标方法相比,PCR方法的符合率为94.3%,敏感性为100%。结论采用溶剂提取制备模板的方法可有效的用于PCR直接检测(无需增菌)乳及乳制品中的金黄色葡萄球菌。
[ Objective ] A uniplex polymerase chain reaction（PCR） assay was developed for direct detection of Staphylococcus aureus without enrichment in dairy products. [ Method ] A solvent extraction procedure was successfully modified for extraction of Staphylococcus aureus DNA from artifically contaminated whole milk, skim milk and cheese. Primer targeting the thermostable nuclease gene （nuc） was used in the uniplex PCR. A DNA fragment of 279 bp was amplified. PCR product was confirmed by DNA sequencing. In this study, the uniplex PCR, GB 4789.10-94, Perifilm RSA. Count Plate, and Baird-parker ＋ RPF Agar were compared. [Result] The detection limit of the uniplex PCR is 10 CFU/ml of whole milk, skim milk and 55 CFU/g cheese. The developed methodology allows detection of Staphylococcus aureus in dairy products in less than 6 h, the time of this developed PCR assay is 12-24 h less than the time of general PCR assay with enrichment method, and the coincidence rates of PCR is 94.3%, the sensitivity of PCR is 100%. [Conclusion] The solvent extraction procedure is an effective method for PCR assay of Staphylococcus aureus in milk and milk products.
出处
《中国农业科学》
CAS
CSCD
北大核心
2006年第5期990-996,共7页
Scientia Agricultura Sinica
基金
河北农业大学科研基金项目(2005-01)
关键词
PCR
检测
乳品
金黄色葡萄球菌
PCR
Detection
Dairy products
Staphylococcus aureus
