志贺氏菌实时荧光单引物等温扩增方法的建立及应用  

Establishment and Application of Real-time Fluorescence Single Primer Isothermal Amplification for Shigella

作　　者：王建昌[1] 胡连霞[1] 段永生[1] 李静[1] 王金凤[1] 

机构地区：[1]河北出入境检验检疫局检验检疫技术中心,河北石家庄050051

出　　处：《食品科学技术学报》2015年第6期40-45,共6页Journal of Food Science and Technology

基　　金：质检公益性科研专项项目(201210128;201310126)

摘　　要：以志贺氏菌ipa H基因特异序列为靶序列,设计RNA-DNA组合引物和链终止序列,优化反应体系,建立实时荧光单引物等温扩增检测志贺氏菌的方法,反应时间为44 min。通过对4株不同群志贺氏菌和12株其他食源性致病菌进行实时荧光单引物等温扩增检测,结果表明,除4株志贺氏菌外,其他细菌均未扩增出荧光曲线。进一步研究表明,采用普通热裂解法提取DNA,实时荧光检测福氏志贺氏菌DNA的灵敏度为1.16 fg/μL,纯培养菌液的灵敏度为1.3 CFU/m L;对牛奶模拟样品中福氏志贺氏菌的检出限是1.8 CFU/m L。研究结果表明,实时荧光单引物等温扩增检测志贺氏菌灵敏度高、特异性强、耗时短、方法简便。Based on the Shigella ipa H gene,the RNA-DNA primers and Blockers were designed and synthetized,and the real-time fluorescence single primer isothermal amplification（ real-time fluorescence SPIA） for the detection of Shigella was established. The reaction time of the method was 44 min. After detection of the 4 different Shigella group strains and 12 other food borne bacteria by the real-time fluorescent SPIA,the results showed that only the Shigella could be detected and showed the typical fluorescence curve. Further studies show that the sensitivity of the detection method for Shigella flexneri in pure culture was 1. 16 fg / μL and 1. 3 CFU / m L. The detection limit of Shigella flexneri in milk was 1. 8 CFU / m L.The results demonstrate that the real-time fluorescence SPIA detection method for Shigella is highly sensitive,strong specific and much more convenient.

关 键 词：实时荧光单引物等温扩增 志贺氏菌 IPAH基因 

分 类 号：TS207.4[轻工技术与工程—食品科学] R117[轻工技术与工程—食品科学与工程]