摘要
建立一种快速检测变形杆菌的方法。采用实时荧光LAMP(Real Amp)技术检测变形杆菌,分析Gen Bank中公布的变形杆菌atp D基因序列,针对其6个区域设计4条引物,利用实时荧光监测仪等温(62℃)扩增模板DNA,通过与电脑相连进行实时监测,对该方法检测变形杆菌的特异性、灵敏度、人工污染猪肉检出限等方面进行研究,并将该检测法的灵敏度与普通环介导等温扩增(LAMP)检测法进行比较。结果表明,Real Amp检测方法比普通LAMP检测方法更加方便快捷,20 min内即可判定结果;用于特异性试验的19株试验菌株中,6株变形杆菌呈阳性结果,其他13株非变形杆菌均呈阴性结果;检测纯菌的灵敏度为8.1CFU/m L,是普通LAMP检测方法的10倍;人工污染猪肉的检出限为81CFU/m L。本研究建立的Real Amp检测方法操作简单,耗时短,特异性强、灵敏度高,能够实现对变形杆菌的快速检测。
We established a rapid detection method for proteus by using real-time fluorescence loop-mediated isothermal amplification （RealAmp） technology. Four loop-mediated isothermal amplification primers were constructed based on the atpD gene of proteus published in GenBank. The portable fluorescence reader （ESE- Quant Tube Scanner） was used for isothermal （62 ℃） amplification of DNA template. Real-time monitoring was achieved by connecting the reader to a computer. Sensitivity, specificity and the detection limit of artificial- ly contaminated pork of proteus were determined using RealAmp and compared with the sensitivity of the con- ventional LAMP. The results showed that RealAmp method was more simple and rapid than the conventional LAMP.The total time of judgment result of the former was generally less than 20 min. A total of 19 strains were subjected to specificity tests, 6 proteus strains indicated positive results, whereas the other 13 non-proteus strains indicated negative results. The sensitivity of RealAmp in detecting pure cultures of proteus was 8.1 CFU/mL, which was10 times compared with that of the conventional LAMP. Moreover, the detection limit of artificially contaminated pork was 81 CFU/mL. The RealAmp method was superior in terms of testing time, convenience, specificity, and sensitivity, and had significant potential for rapid detection of proteus.
出处
《食品研究与开发》
CAS
北大核心
2016年第11期127-132,共6页
Food Research and Development
基金
河北省自然科学基金项目(C2008000216)
