摘要
为快速检测污染鸡蛋的致病菌,实验从鸡蛋蛋体表面得到2株菌X1、X2,结合16S r RNA克隆及质粒测序分子方法对2株菌分类分析,发现2株菌X1和X2分别为:蜡样芽孢杆菌Bacillus cereus NC7401(AP007209)、金黄色葡萄球菌Staphylococcus aureus(AP003088)。针对两种菌的nhe、nuc基因具有较强的保守性,设计2套环介导等温扩增(LAMP)引物。先进行反应体系优化,得到25μL LAMP反应体系最适温度为60℃、最适Mg SO4添加量为1.5μL,引物最适量为2.0μL。再进行二重LAMP鉴定2株污染鸡蛋的致病菌,发现目的菌X1和X2的特异性扩增结果为阳性,其他9株非目的菌扩增结果为阴性,其灵敏度检测限达10 fg/μL。此二重LAMP检测法有快速、特异、灵敏的特点,可快速检测食源性致病菌。
Two strains of bacteria named X_1,X_2 were found to detect the pathogenic bacteria on eggs. Combined with 16 S r RNA molecule methods,testing the cloned plasmid sequence,blasting the sequence with NCBI in Gen Bank. The results showed that X_1 was Bacillus cereus NC7401（AP007209） and X_1 was Staphylococcus aureus（AP003088）. According to the highly conservative gene nhe and nuc,two sets of primers were designed.The optimized LAMP reaction system contained Mg SO41.5 μL,each primer 2.0 μL and reaction temperature of60 ℃. The specificity was verified by using 9 strains of non-targeting bacteria and targeting bacteria X_1,X_2.The LAMP assay showed a sensitivity of 10 fg / μL. The novel multiple LAMP method reported here was characteristics of rapid detection, high specificity and excellent sensitivity, which could be applied in the detection of pathogenic bacteria.
出处
《食品工业科技》
CAS
CSCD
北大核心
2016年第4期107-110,116,共5页
Science and Technology of Food Industry
