基于SYBR Green Ⅰ荧光定量PCR建立生乳及乳制品沙门氏菌快速检测技术  

SYBR Green Ⅰ real-time polymerase chain reaction for rapid detection of Salmonella spp. in raw milk and milk products

作　　者：张巧艳[1] 陈亭亭[2] 陈笑芸[1] 杨胜利[2] 缪青梅[1] 

机构地区：[1]浙江省农业科学院农产品质量标准研究所,浙江杭州310021 [2]浙江工业大学药学院,浙江杭州310014

出　　处：《浙江农业学报》2012年第5期914-921,共8页Acta Agriculturae Zhejiangensis

基　　金：浙江省公益技术应用研究项目(2010C32024,2012C22069);农业部948项目(2011G5(2))

摘　　要：生乳易受沙门氏菌污染,并大量繁殖;经加工的乳制品细菌数大大减少,但仍存在沙门氏菌污染的风险。试验按生乳及乳制品沙门氏菌污染水平研究样品前处理方法,采用煮沸裂解法快速提取总DNA,针对沙门氏菌invA基因建立SYBR Green Ⅰ荧光定量PCR快速检测技术。建立的方法具有良好的特异性和较高的灵敏度,其中生乳沙门氏菌检出限为102cfu.mL-1,方法的检测线性范围为102～108cfu.mL-1,相关系数(R2)为0.999,扩增效率为103%;乳制品沙门氏菌经16 h增菌后检出限达到1 cfu.[25 g(mL)]-1。该技术可用于生乳中沙门氏菌的高效筛查及定量检测,检测一个样品仅需3 h;同时,可用于乳制品的准确定性检测,检测周期为1 d;且方法重复性好、准确度高、操作简单,为乳制品企业快速检测沙门氏菌提供了有效的技术手段。Raw milk was susceptible to Salmonella spp. , which was reproduced rapidly. Nevertheless, there was only the potential of very small amount of Salmonella spp. in milk products since sterilization and other treatments though the bacterial population decreased. In this paper, we studied the pretreatment methods of raw milk and milk products according to their contamination levels and optimized the extraction conditions of bacterial total DNA by the boiling lysis procedure. Furthermore, we set up a systemic detection technology for milk-borne Salmonella spp. , based on SYBR Green I real-time PCR targeting the invA gene. The developed methods possessed specificity for SalmoneUa spp. As far as raw milk was concerned, the limit of detection of the method was 102 cfu.mL-1, the linear range of the standard curve was 102 - 10s cfu-mL-l, correlation coefficient（R2） was 0. 999, and amplification effi- ciency was 103%. For milk products, 1 cfu of Salmonella spp. present in 25 g （mL） milk samples was shown de- tectable by this method with an enrichment step of 16 h. This technology could be used to quantitatively detect Sal-monella spp. in raw milk only for 3 h, and qualitatively assay milk products only for 1 d. All in all, our work provid- ed a high-throughout detection technology of .Salmonella spp. for dairy enterprises, which was proved rapid, simple, sensitive and accurate with a good reproducibility.

关 键 词：沙门氏菌 荧光定量PCR SYBR Green Ⅰ DNA提取 生乳 乳制品 

分 类 号：R155.57[医药卫生—营养与食品卫生学]