荧光定量 PCR 检测原料乳中鲁氏不动杆菌方法的建立

张慧敏; 夏海磊; 王梦琦; 邢世宇; 毛永江; 岑宁; 杨章平

doi:10.13386/j.issn1002-0306.2017.03.051

荧光定量 PCR 检测原料乳中鲁氏不动杆菌方法的建立

Study on fluorogenic quantitative PCR detection method of Acinetobacter lwoffii in raw milk

摘要

摘要: 为建立鲁氏不动杆菌（Acinetobacter lwoffii）的快速检测方法,本研究以A.lwoffii中的β-内酰胺酶基因(ALbla_OXA-134)为目的基因设计特异性引物,建立了SYBR GreenⅠ实时荧光定量PCR检测A.lwoffii的方法。结果显示,该方法可快速检测A.lwoffii的存在,检测灵敏度为2.4×10²cfu/m L,建立的标准曲线为y=-3.838x+40.93,相关系数为0.993。与其他原料乳中常见的微生物均未发现交叉反应。人工阳性样品乳中A.lwoffii的检测限为2.4×10³cfu/m L。最后,对2015年11月至2016年1月长江下游地区8个牧场的原料乳进行监测。结果表明该检测方法具有较好的敏感性、特异性和实用性,为快速检测原料乳中的A.lwoffii提供了有力的技术支撑,有利于保障原料乳的质量安全。

Abstract: In order to detect the Acinetobacter lwoffii quickly and accurately,a real-time PCR detection based on SYBR GreenⅠ was established with a pair of primers designed according to the β-lactamase gene of A.lwoffii( AL-bla_(OXA-134)).The result showed that this method could detect the A.lwoffii quickly in raw milk with a detection limit of 2.4 × 10~2 cfu/m L,and without any cross-reaction to other microorganisms in raw milk. The standard curve was y =-3.838 x + 40.93,of which correlation coefficient was 0.993. The detection limit of A. lwoffii in artificial contaminated milk sample was 2.4 × 10~3 cfu/m L by this method.Finally,milk sample collected from eight pastures in lower reaches of the Yangtze River since Nov.,2015 to Jan.,2016 was analyzed by this method.The method established in this study was specific,highly sensitive,which could be further used in detection of A.lwoffii in raw milk and helpful to improve the quality safety of raw milk.