Abstract Listeria monocytogenes is significantly associated with listeriosis infection. Several species of Listeria other than L. monocytogenes directly affect the food industry. In the present study we developed a novel subtyping method for tracking the source of L. monocytogenes and L. innocua in large-scale investigations using multiple-locus variable-number tandem repeat analysis (MLVA) coupled with next-generation sequencing (NGS). Forty-eight L. monocytogenes and L. innocua strains isolated from a food processing plant and the environment were used for the amplification of 15 specific variable number tandem repeat (VNTR) loci. The results exhibited 100% concordance for amplicon detection using both the subtyping methods. Coupling of MLVA with capillary electrophoresis (CE) or NGS detected 38 and 89 different alleles, respectively. MLVA coupled with NGS showed significantly higher discriminating power than MLVA coupled with CE. In MLVA coupled with NGS method, locus JLR1 of L. monocytogenes had 54% discrimination while locus TR3 had 100% discrimination solely in L. innocua. In addition, MLVA coupled with NGS method had significantly higher discrimination power (DI = 1.0 with 48 MLVA patterns) than that of MLVA coupled with CE (DI = 0.79 with 15 MLVA patterns). These results indicate the advantage of MLVA coupled with NGS method in detecting not only the length but also sequence polymorphism in a single read run. MLVA coupled with NGS led to significant reduction in labor and cost than CE and the traditional Sanger sequencing. Thus, MLVA coupled with NGS is an effective and feasible technique for the molecular typing of Listeria species in food industries.

中文翻译：

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使用多重聚合酶链反应和下一代测序进行多位点可变数量串联重复分析——一种新型的高通量李斯特菌菌株亚型方法

摘要 单核细胞增生李斯特菌与李斯特菌病感染显着相关。除单核细胞增生李斯特菌外的几种李斯特菌直接影响食品工业。在本研究中，我们开发了一种新的亚型分型方法，用于在使用多基因座可变数量串联重复分析 (MLVA) 和下一代测序 (NGS) 的大规模调查中跟踪单核细胞增生李斯特氏菌和无害李斯特氏菌的来源。从食品加工厂和环境中分离出的 48 株单核细胞增生李斯特菌和无害李斯特菌菌株用于扩增 15 个特定的可变数串联重复 (VNTR) 基因座。使用两种亚型方法检测扩增子的结果均显示 100% 的一致性。MLVA 与毛细管电泳 (CE) 或 NGS 的偶联分别检测到 38 个和 89 个不同的等位基因。MLVA 与 NGS 结合显示出比 MLVA 与 CE 结合显着更高的辨别力。在MLVA结合NGS方法中，单核细胞增生李斯特菌的基因座JLR1具有54%的辨别力，而基因座TR3仅在无害李斯特氏菌中具有100%的辨别力。此外，MLVA 与 NGS 方法结合的鉴别能力（DI = 1.0，48 个 MLVA 模式）比 MLVA 与 CE 结合（DI = 0.79，15 个 MLVA 模式）具有显着更高的鉴别能力。这些结果表明 MLVA 与 NGS 方法结合在单次读取运行中不仅检测长度而且检测序列多态性的优势。与 CE 和传统的 Sanger 测序相比，MLVA 与 NGS 相结合显着降低了劳动力和成本。因此，MLVA 与 NGS 结合是食品工业中李斯特菌物种分子分型的一种有效且可行的技术。