Development of a Detection Method for Assaying Calf Intestine Alkaline Phosphatase Activity

[ Objective] This study was to develop a detection method for assaying calf intestine alkaline phasphatase activity in scientific research. [ Method ] By simulating the conditions and buffers for assaying calf intestine alkaline phosphatase used in the scientific research, the parameters influencing substmte concentration, reaction duration, eoloration time and buffer pH were optimized. [ Result] The activity of alkaline phosphatase detected varied hugely among different buffers, and potassium ferricyanide solution added with boracic acid achieved the stable coloration. The optimal water bath time was determined as 10 rain, and the substrate concentration was optimized as 0.04 moL/L. The increasing temperature did not have a large influence on low temperature enzymatic activity while did on high coneentration enzymatic activity. When the buffer pH was 7.0 - 8.0, the detection stability of alkaline phosphatase could be maintained well The ion intensity of buff- er had little effects on alkaline phasphatase activity. [ Conclusion] A detection method for assaying calf intestine alkaline phosphatase activity in scientific research was successfully developed in this study.

食品中蜡样芽胞杆菌的毒力基因分布研究

为了调查食品中蜡样芽胞杆菌的污染状况,以及蜡样芽胞杆菌毒力基因的分布情况,采集了419份食物样本,从样本中分离出32株蜡样芽胞杆菌,检出率为7.6%。应用hblA、hblC、hblD三重基因,nheA、nheB、nheC三重基因,entFM、bceT、ces三重基因的实时荧光PCR体系对阳性样本进行毒力基因分布的研究。结果表明,hblA、hblC、hblD基因检出率均为53.1%;nheA、nheB、nheC基因检出率分别为65.6%、90.6%、87.5%;entFM和bceT基因检出率分别为96.9%和53.1%,携带ces基因的仅有一株。除ces基因外,携带上述八种基因的菌株占阳性菌株的34.4%。

侧向流动型重组酶聚合酶扩增技术检测食品中大肠埃希氏菌O157

研究内容基于侧向流动型重组酶聚合酶扩增(Lateral flow-based recombinase polymerase amplification,LF-RPA)技术原理,建立大肠埃希氏菌O157(Escherichia coli O157)的LF-RPA快速检测方法。应用该方法对3株大肠埃希氏菌O157和27株非大肠埃希氏菌O157进行检测,结果表明该方法具有良好的特异性。分别以10倍浓度梯度稀释插入靶基因序列的质粒pUC57-rfbE和大肠埃希氏菌O157菌液DNA作为模板进行LF-RPA检测。结果表明LF-RPA方法可检出1.0×10^(2)拷贝/μL的pUC57-rfbE质粒和1.2×10^(4) cfu/mL的大肠埃希氏菌O157菌液。以大肠埃希氏菌O157污染食品样品,增菌4 h后,大肠埃希氏菌O157初始浓度为1.2×10^(1) cfu/mL的食品增菌液可以被LF-RPA检出阳性信号。该研究建立的方法可快速、准确、灵敏地检测食品中大肠埃希氏菌O157,无需专业设备,在食品生产企业和基层检测机构具有一定的应用前景。

三氯羟基二苯醚中和剂筛选及杀菌效果研究

目的为含三氯羟基二苯醚(三氯生)的消毒剂选择合适的中和剂,观察不同浓度三氯生消毒剂的杀菌效果。方法应用载体定量鉴定试验方法筛选中和剂,应用载体定量杀菌试验方法观察杀菌效果。结果中和剂1(含2.0%吐温80、2.0%卵磷脂的磷酸盐缓冲液)能有效中和残留的消毒剂A(0.35%三氯生+40%乙醇+10%丙二醇)、消毒剂B(0.5%三氯生+50%乙醇)和消毒剂C(0.1%三氯生+5%乙醇),且中和剂和中和产物不影响试验菌的生长。中和剂2(含0.5%甘氨酸、1.0%吐温80、1.0%卵磷脂的磷酸盐缓冲液)和中和剂3(含1.0%组氨酸、3.0%吐温80、0.5%卵磷脂的磷酸盐缓冲液)不能中和消毒剂A。消毒剂B、C对金黄色葡萄球菌作用1 min的平均杀灭对数值>3.00,消毒剂C对大肠埃希菌、白色念珠菌作用5 min的平均杀灭对数值>3.00。结论中和剂1是三氯生消毒剂的合格中和剂,三氯生消毒剂具有较好的杀菌效果。