School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China; Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China.
Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China.
Int J Food Microbiol. 2021 Feb 2;339:109026. doi: 10.1016/j.ijfoodmicro.2020.109026. Epub 2020 Dec 16.
The abundant information provided by the pan-genome analysis approach reveals the diversity among Listeria monocytogenes serotypes. The objective of this study was to mine novel target genes using pan-genome analysis for multiplex PCR detection and differentiation of the major L. monocytogenes serotypes present in food. Pan-genome analysis and PCR validation revealed a total of 10 specific targets: one for lineage I, two for serogroup I.1, one for serogroup I.2, two for lineage II, one for serogroup II.1, three for lineage III. Primers for the novel targets were highly specific in individual reactions. The detection limits were 10-10 colony-forming units (CFU)/mL in pure bacterial cultures, meeting the requirements of molecular detection. Based on these novel targets, two new "lineage" multiplex PCR assays were developed to simultaneously distinguish between three lineages (I, II, and III) and five major serotypes (1/2a, 1/2b, 1/2c, 4b, and 4c) of L. monocytogenes. The detection limits of lineage I and lineage II&III mPCRs were 0.771 pg/μL and 1.76 pg/μL genomic DNA, respectively. The specificity of the mPCRs was robustly verified using other L. monocytogenes and non-L. monocytogenes serotypes. These results suggest that the two "lineage" multiplex PCRs based on novel targets offer a promising approach for accurate, sensitive, and rapid identification of L. monocytogenes serotypes.
泛基因组分析方法提供的丰富信息揭示了单核细胞增生李斯特菌血清型之间的多样性。本研究旨在利用泛基因组分析挖掘新的靶基因，用于多重 PCR 检测和区分食品中存在的主要单核细胞增生李斯特菌血清型。泛基因组分析和 PCR 验证共揭示了 10 个特定靶标：1 个用于谱系 I，2 个用于血清群 I.1，1 个用于血清群 I.2，2 个用于谱系 II，1 个用于血清群 II.1，3 个用于谱系 III。针对这些新靶标的引物在单个反应中具有高度特异性。在纯细菌培养物中的检测限为 10-10 菌落形成单位 (CFU)/mL，满足分子检测的要求。基于这些新靶标，开发了两种新的“谱系”多重 PCR 检测方法，可同时区分三个谱系 (I、II 和 III) 和单核细胞增生李斯特菌的五个主要血清型 (1/2a、1/2b、1/2c、4b 和 4c)。谱系 I 和谱系 II&III mPCR 的检测限分别为 0.771 pg/μL 和 1.76 pg/μL 基因组 DNA。使用其他单核细胞增生李斯特菌和非单核细胞增生李斯特菌血清型对 mPCR 的特异性进行了稳健验证。这些结果表明，基于新靶标的两种“谱系”多重 PCR 为准确、敏感、快速鉴定单核细胞增生李斯特菌血清型提供了一种有前途的方法。
