酸乳中葡糖杆菌的LAMP检测方法研究

Study on LAMP for Detecting Gluconobacter in Yogurt

为了给酸乳生产企业提供一种快捷、简便、灵敏的葡糖杆菌的LAMP检测方法,本研究选取葡糖杆菌属的模式菌株氧化葡糖杆菌为代表菌株。根据Gen Bank中公布的氧化葡糖杆菌的16S r RNA(X73820)基因的强特异性序列设计四条引物,优化引物、Mg2+、温度等反应条件,建立了环介导等温扩增技术检测酸乳中葡糖杆菌的方法。对该方法的特异性和灵敏度进行评价,并与PCR方法的灵敏度进行比较。结果显示,该组引物的特异性强,对反应产物进行酶切分析,酶切产物的片段与理论值相符;该LAMP方法检测纯菌的灵敏度为7.5×101 CFU/m L,是PCR检测方法的10倍;用纯菌液对酸奶进行人工污染,提取模拟变质酸奶的DNA进行LAMP扩增,其最低检出限为7.5×102 CFU/m L。综上所述,本研究建立的LAMP检测方法特异性强、灵敏度高,耗时短,实现了对葡糖杆菌的快速检测,在食品检测行业具有很好的应用前景。

Objective： The aim was to develop a rapid, simple, and sensitive LAMP method for the detection of Gluconobacter to be used in the yogurt industry. Method： Gluconobacter oxydans was selected as the type culture strain. Two pairs of LAMP primers targeting the 16 S r RNA（X73820）housekeeping gene of Gluconobacter available in Gen Bank were designed. Concentrations of the primers and Mg2＋ and the temperature of the LAMP were optimized, and a rapid, sensitive LAMP assay for Gluconobacter detection was established. Sensitivity of the assay was evaluated and compared with the PCR assay. Results： It was found that the LAMP primers showed high specificity and the LAMP product sizes, after restriction enzyme digestion, were consistent with the theoretical fragment sizes. The sensitivity of the LAMP assay for Gluconobacter was 7.5 × 101 CFU/m L. The LAMP assay was found to be 10-fold more sensitive than the traditional PCR assay. The LAMP detection limit was 7.5 × 102 CFU/m L for artificial contaminated yogurt. Conclusion： LAMP amplification established in this study provides a rapid,highly specific and sensitive method for Gluconobacter detection. The LAMP method is valuable for its application and exploitation in food industries.