Takahashi Hajime, Handa-Miya Satoko, Kimura Bon, Sato Miki, Yokoi Asami, Goto Seitaro, Watanabe Itaru, Koda Takashi, Hisa Kazuo, Fujii Tateo
Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
Int J Food Microbiol. 2007 Sep 30;118(3):274-84. doi: 10.1016/j.ijfoodmicro.2007.07.047. Epub 2007 Aug 7.
Development of rapid and simple typing methods is required for analyzing the distribution and contamination routes of food-borne pathogens. We established a simple typing method for Listeria monocytogenes using MLSSCP (Multilocus Single Strand Conformation Polymorphism) analysis. Four virulence genes, hlyA, iap, actA and inlB were amplified by PCR, digested with endonucleases and applied to gels for SSCP. As banding patterns have been shown to reflect even a single nucleotide difference, this method has a potential discriminatory power comparable to that of sequencing analysis. The 64 strains isolated from five meat processing plants were divided into 18 groups by this MLSSCP. Additionally, clustering obtained with this method showed strong correspondence with phylogenetic lineages I and II, and was achieved with much less expenditure in time and cost than is required for other methods, such as MLST. The validity of the MLSSCP lineage classification was confirmed by PFGE, AFLP and ribotyping results. This newly developed MLSSCP method is suitable when obtaining accurate results quickly and simply is crucial.
为了分析食源性病原体的分布和污染途径，需要开发快速简便的分型方法。我们利用多位点单链构象多态性（MLSSCP）分析建立了一种简单的单核细胞增生李斯特菌分型方法。通过PCR扩增四个毒力基因hlyA、iap、actA和inlB，用核酸内切酶消化后应用于凝胶进行SSCP分析。由于条带模式已被证明能反映即使是单个核苷酸的差异，该方法具有与测序分析相当的潜在鉴别力。通过这种MLSSCP方法，从五个肉类加工厂分离出的64株菌株被分为18组。此外，用该方法获得的聚类结果与系统发育谱系I和II有很强的对应关系，并且与其他方法（如MLST）相比，在时间和成本上的花费要少得多。PFGE、AFLP和核糖体分型结果证实了MLSSCP谱系分类的有效性。当快速简单地获得准确结果至关重要时，这种新开发的MLSSCP方法是合适的。
