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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 is susceptible to Salmonella contamination and multiplies; there is a significant reduction in the number of bacteria in processed dairy products, but there is still a risk of Salmonella contamination. The method of sample preparation was studied according to the contamination level of Salmonella in raw milk and dairy products, and the total DNA was extracted rapidly by boiling pyrolysis. The rapid detection of SYBR Green Ⅰ fluorescence quantitative PCR for Salmonella invA gene was established. The established method has good specificity and high sensitivity, with the detection limit of 102 cfu.mL-1 for S.pastoris and the detection linear range of 102-108 cfu.mL-1 and the correlation coefficient (R2) of 0.999 The efficiency was 103%. The detection limit of dairy products Salmonella reached 1 cfu after 16 h enrichment [25 g (mL)] - 1. The technology can be used for the efficient screening and quantitative detection of Salmonella in raw milk. It takes only 3 hours to test one sample. Meanwhile, it can be used for the accurate qualitative detection of dairy products with a detection period of 1 day. The method has good repeatability, high accuracy, Simple operation, for the rapid detection of Salmonella dairy enterprises to provide an effective technical means.