Xu Yun-Ming, Liu Xi-Lin, Ma Jing, Li Yan-Song, Hu Pan, Zou De-Ying, Guo Xing, Chen Xiao-Feng, Tang Feng, Liu Nan-Nan, Wei Li-Bin, Zhou Yu, Liu Zeng-Shan, Ren Hong-Lin, Lu Shi-Ying
Southeast Asian J Trop Med Public Health. 2014 May;45(3):670-9.
Yersinia enterocolitica (YE) is a main pathogenic bacterium causing diarrhea and yersiniosis occurs in both developed and developing countries with high incidence. YE in contaminated food is able to survive for a long duration even under cold storage, thereby enhancing the risk of food infection. In this study, a new loop-mediated isothermal amplification (LAMP) method showing the characteristics of simplicity, rapidity, high specificity and sensitivity was established by targeting outL of pathogenic YE. Two inner-primers and outer-primers were designed and LAMP reaction was optimized for Mg2+, betaine, dNTPs and inner primers concentrations, reaction temperature and time. Sensitivity and specificity of the LAMP assay was evaluated using YE genomic DNA and those of 44 different bacteria strains, respectively. Validation of LAMP detection method was by employing meat samples spiked with varying CFU of YE. The optimized LAMP assay was specific, capable of detecting 97 fg of genomic DNA (equivalent to 37 genome copies) of YE (100-fold more sensitive than PCR) and 80 CFU/ml of YE-spiked meat samples based on ethidium bromide stained amplicon bands on agarose gel-electrophoresis and on GelRed fluorescence of the LAMP reaction solution, respectively. This rapid, sensitive and specific LAMP technique should enable application in field inspection of Y. enterocolitica in food.
小肠结肠炎耶尔森菌（YE）是引起腹泻的主要病原菌，耶尔森菌病在发达国家和发展中国家的发病率都很高。受污染食品中的YE即使在冷藏条件下也能长期存活，从而增加了食物感染的风险。在本研究中，通过靶向致病性YE的outL基因，建立了一种具有简单、快速、高特异性和高灵敏度特点的新型环介导等温扩增（LAMP）方法。设计了两条内引物和外引物，并对LAMP反应的Mg2+、甜菜碱、dNTPs和内引物浓度、反应温度和时间进行了优化。分别使用YE基因组DNA和44种不同细菌菌株评估了LAMP检测方法的灵敏度和特异性。通过使用添加了不同CFU的YE的肉类样品对LAMP检测方法进行验证。优化后的LAMP检测方法具有特异性，基于琼脂糖凝胶电泳上溴化乙锭染色的扩增条带和LAMP反应溶液的GelRed荧光，分别能够检测到97 fg的YE基因组DNA（相当于37个基因组拷贝）（比PCR灵敏100倍）和80 CFU/ml添加YE的肉类样品。这种快速、灵敏且特异的LAMP技术应能应用于食品中肠炎耶尔森菌的现场检测。
