A rapid typing method for Listeria monocytogenes based on high-throughput multilocus sequence typing (Hi-MLST)

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• 作者：Hajime Takahashi ; ^{hajime@kaiyodai.ac.jp} ; Ai Iwakawa ; Chihiro Ohshima ; Daisuke Kyoui ; Shiori Kumano ; Takashi Kuda ; Bon Kimura
• 关键词：Listeria monocytogenes ; Molecular typing ; Next generation sequencing ; Multilocus sequence typing
• 刊名：International Journal of Food Microbiology
• 出版年：2017
• 出版时间：21 February 2017
• 年：2017
• 卷：243
• 期：Complete
• 页码：84-89
• 全文大小：583 K
• 卷排序：243

文摘

Listeria monocytogenes infects humans via food products, causing listeriosis. Consequently, food companies pay meticulous attention to the risk of contamination of their products by this bacterium. While fragment analysis methods such as pulsed-field gel electrophoresis (PFGE) are used to trace the sources of contamination for this bacterium, some drawbacks have been identified, namely the complexity of the methods and the difficulty of making data comparisons. As an alternative, multilocus sequence typing (MLST) is now seeing widespread use; however, owing to its cost, time, and labor requirements, its diffusion into the food industry has been slow. Thus, in the present study, a High-throughput MLST (Hi-MLST) method, which can rapidly, simply, and cheaply perform MLST analyses using a next-generation sequencer (NGS) that can analyze a large volume of base sequences at once was developed. Firstly, a multiplex PCR method designed to amplify seven genes for use in MLST was developed. The discriminatory potential of the developed method was confirmed in silico, and was verified that it has the same discriminatory potential as conventional methods. Next, MLST analysis using multiplex PCR and NGS was performed for 48 strains of L. monocytogenes. The sequences obtained from this analysis have sufficiently reliable quality for all of the genes from of all the strains. Thus, this method could classify the 48 strains into 39 sequence types (ST) with a Diversity index (DI) of 0.989. In summary, using the Hi-MLST method developed in the present study, which combined multiplex PCR and NGS, cut the costs to 1/6th and the time to 1/20th that of conventional MLST methods.