Keto-Timonen Riikka O, Autio Tiina J, Korkeala Hannu J
Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Finland.
Syst Appl Microbiol. 2003 Jun;26(2):236-44. doi: 10.1078/072320203322346083.
Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.
最初对9种限制性内切酶组合和108种不同的引物组合进行了测试，以确定其是否适合用于单核细胞增生李斯特菌的扩增片段长度多态性（AFLP）分析；HindIII和HpyCH4IV的组合在凝胶上始终显示出强烈的信号，扩增出足够数量的DNA片段，并根据AscI酶切图谱检测出密切相关菌株之间的多态性。AFLP还能区分单核细胞增生李斯特菌、无害李斯特菌、伊氏李斯特菌、斯氏李斯特菌、威氏李斯特菌和格氏李斯特菌。所有李斯特菌属物种都显示出物种特异性聚类，不同物种之间的相似度低于33%。总共使用AFLP和脉冲场凝胶电泳（PFGE）对34株单核细胞增生李斯特菌进行了鉴定。单核细胞增生李斯特菌菌株的AFLP分析结果与PFGE获得的结果一致。两种方法都鉴定出29种不同的单核细胞增生李斯特菌基因型，并且具有较高的鉴别指数（>0.999）。通过结合AFLP和PFGE的结果，进一步提高了亚型鉴别能力。对AFLP和PFGE图谱的数值分析产生了单核细胞增生李斯特菌菌株的三个基因组群。发现AFLP比PFGE更快且劳动强度更低。我们得出结论，AFLP方案是一种用于李斯特菌菌株鉴定的高度鉴别性、可重复且有价值的工具，也可能适用于李斯特菌属物种的鉴定。
