利用多重 PCR 检测法检测鲜切叶类蔬菜中的节杆菌属和醋酸钙不动杆菌。
Detection of Exiguobacterium spp. and E. acetylicum on fresh-cut leafy vegetables by a multiplex PCR assay.
机构信息
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China.
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China.
出版信息
J Microbiol Methods. 2021 Jan;180:106100. doi: 10.1016/j.mimet.2020.106100. Epub 2020 Nov 27.
AIMS
To identify the main spoilage bacterium on fresh-cut leafy vegetables and establish a multiplex PCR assay.
METHODS AND RESULTS
Based on physiological-biochemical, molecular identification, and artificial contamination tests, the main bacterium to spoil fresh-cut leafy vegetables was identified as Exiguobacterium spp. and Exiguobacterium acetylicum. Comparative genomics showed that P401_RS0117025 and oxi_50,582,462 genes are specific to Exiguobacterium spp. and E. acetylicum. Based on this, three pairs of primer sets to EaG-291, EaS-2B, and Ea16S-12 genes were designed and used to develop a multiplex PCR assay, which exhibited 100% specificity among 16 Exiguobacterium and 10 non-Exiguobacterium strains. Finally, 84 fresh-cut leafy vegetable samples were analyzed by multiplex PCR assay and standard physiological-biochemical experiments, the results showed multiplex PCR assay reached a detection rate of 96%.
CONCLUSIONS
The main spoilage bacterium was identified as Exiguobacterium spp. and E. acetylicum on fresh-cut leafy vegetables based on the novel specific genes explored in this study.
SIGNIFICANCE AND IMPACT OF STUDY
A rapid, specific, and sensitive PCR assay was developed for the detection of Exiguobacterium spp. and E. acetylicum.
目的
鉴定鲜切叶类蔬菜中的主要腐败菌，并建立多重 PCR 检测方法。
方法与结果
基于生理生化、分子鉴定和人工污染试验，确定鲜切叶类蔬菜的主要腐败菌为节杆菌属和醋酸钙不动杆菌。比较基因组学表明，P401_RS0117025 和 oxi_50、582、462 基因是节杆菌属和醋酸钙不动杆菌特有的。基于此，设计了三对针对 EaG-291、EaS-2B 和 Ea16S-12 基因的引物对，建立了多重 PCR 检测方法，该方法在 16 株节杆菌和 10 株非节杆菌菌株中具有 100%的特异性。最后，用多重 PCR 检测方法和标准生理生化实验对 84 份鲜切叶类蔬菜样品进行了分析，结果表明，该方法的检测率达到 96%。
结论
基于本研究中探索的新的特异性基因，鉴定出鲜切叶类蔬菜中的主要腐败菌为节杆菌属和醋酸钙不动杆菌。
研究意义与影响
建立了一种快速、特异、敏感的检测节杆菌属和醋酸钙不动杆菌的 PCR 检测方法。
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