根据假单胞菌16S rDNA基因序列设计引物,并向反应体系中加入饱和型核酸染料Eva Green,利用实时荧光检测仪,建立假单胞菌属实时荧光环介导等温扩增技术(LAMP)。研究中对62株假单胞菌的16SrDNA基因通过DNAMAN软件进行序列比对后,针对共有片段设计扩增引物。对扩增体系进行了优化,优化结果经电泳鉴定。通过12株假单胞菌和23株非假单胞菌进行特异性验证,并比较了实时荧光LAMP与普通LAMP的灵敏度,对人工污染样品的检出限进行了测定。结果表明,特异性验证中12株假单胞菌为阳性,23株非假单胞菌为阴性。实时荧光LAMP检测纯菌的灵敏度为36 CFU/mL,是普通LAMP灵敏度的10倍。人工污染样品的检出限为1.73×10^3 CFU/g。本研究中建立了一种检测冷鲜猪肉中假单胞菌属的方法,实现了多个菌种的同时检测,避免了单一菌种检测的局限性,该方法快速、准确、灵敏度高,可在2h内完成冷鲜猪肉中假单胞菌的检测。
In this work, a real-time fluorescence loop-mediated isothermal amplification (LAMP) assay was established to detect Pseudomonas in pork. Based on the published 16S rDNA gene sequence, the 16S rDNA genes of 62 strains of Pseudomonas were sequenced by DNAMAN software to obtain the consensus fragment, which was used to design primers. The saturated nucleic acid dye Eva Green was added to the reaction system, which allowed amplification products to be measured by real-time fluorescence platforms. The amplification system was optimized and the optimization results were identified by electrophoresis. The specificity and sensitivity was compared with ordinary LAMP, and the detection limit of artificially contaminated pork was determined. The results showed that 12 strains of Pseudomonas are positive in the specific verification and 23 strains of non-Pseudomonas are negative. The sensitivity for Pseudomonas is 36 CFU/mL in pure cultures, which is 10 times more sensitive than the ordinary LAMP. The detection limit of artificially contaminated samples is 1.73×10^3 CFU/g. In this work, a method for detecting Pseudomonas in refrigerated pork was established, which can simultaneously detect Pseudomonas spp. and avoid the limitations of single species detection. The real-time fluorescent LAMP detection technology established in this work is less time-consuming, accurate and has high specificity and sensitivity. Pseudomonas in fresh pork could be detected within 2 hours by this method.
Modern Food Science and Technology
real-time fluorescent loop-mediated isothermal amplification