摘要
建立一种荧光染料叠氮溴化丙锭(PMA)与环介导等温扩增(LAMP)相结合的技术,同时利用羟基萘酚蓝(HNB)染料对婴儿配方奶粉中沙门氏菌活菌进行快速可视化检测。以沙门氏菌siiA基因为靶点,设计特异性引物,利用PMA抑制死菌DNA的扩增反应,反应前向体系中加入HNB染料,通过颜色变化对目标菌进行快速检测。结果表明,PMA的最佳处理浓度为3μg/mL,体系中HNB的最优浓度为150μmol/L,所构建的PMA-LAMP-HNB方法对沙门氏菌纯培养物的检测灵敏度为4.6×10^1CFU/mL,对人工污染婴儿配方奶粉中该菌的检测灵敏度为6.3×10^1CFU/g,是传统PCR方法的100倍,且总检测时间不超过1 h 30 min。
In order to realize rapid and visual detection of viable Salmonella,a new method was established by combination of propidium monoazide(PMA)and loop-mediated isothermal amplification(LAMP),which was simultaneously utilized with hydroxy naphthol blue(HNB).Specific primers were designed based on siiA gene,and PMA was used to inhibit DNA amplification of dead bacteria.At the same time,HNB dye was pre-added to LAMP system and the target bacteria were rapidly detected by color changing.The results showed optimal concentration of PMA and HNB was 3μg/mL and 150μmol/L,respectively.The detection sensitivity of Salmonella in pure cultures was 4.6×10~1 CFU/mL,while that of 6.3×10~1 CFU/g in artificial contaminated powdered infant formula,which was 100 times than that of traditional PCR method.Meanwhile,the total detection time can be controlled in 1 h 30 min.
作者
王冠蕾
WANG Guanlei(Chengde Petroleum College,Chengde 067000,China)
出处
《中国乳品工业》
CAS
北大核心
2020年第4期51-54,64,共5页
China Dairy Industry
关键词
沙门氏菌
叠氮溴化丙锭
环介导等温扩增
羟基萘酚蓝
婴儿配方奶粉
Salmonella
Propidium monoazide
Loop-mediated isothermal amplification
Hydroxy naphthol blue
Powdered infant formula
