Somer Lilach, Danin-Poleg Yael, Diamant Eran, Gur-Arie Riva, Palti Yniv, Kashi Yechezkel
Department of Biotechnology and Food Engineering, Technion--Israel Institute of Technology, Haifa 32000, Israel.
Appl Environ Microbiol. 2005 Jun;71(6):3144-52. doi: 10.1128/AEM.71.6.3144-3152.2005.
DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of Listeria monocytogenes and 6 strains of Listeria spp. and 30 AILP markers among 27 strains of Escherichia coli. Up to four alleles per locus were identified among Listeria strains, and up to six were identified among E. coli strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among Listeria and E. coli strains. A clear distinction between L. monocytogenes and Listeria spp. was demonstrated. In addition, the method separated L. monocytogenes into the three known lineages and discriminated the most common virulent serotypic group, 4b. In E. coli, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both Listeria and E. coli are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.
基于DNA的方法在细菌分型中越来越重要。密切相关的细菌基因组中存在大量多态性位点，这是本文所述方法的基础。该方法可识别基因间区域的多位点基因组多态性，称为AILP（扩增基因间位点多态性）。对于每个位点，设计一对独特的PCR引物，以扩增从一个开放阅读框（ORF）到相邻ORF的基因间序列。鉴定扩增产物的存在、缺失和大小变异，并将其用作快速区分菌株的遗传标记。使用单核细胞增生李斯特菌28株、李斯特菌属6株中的18个AILP位点以及大肠杆菌27株中的30个AILP标记评估多态性。在李斯特菌菌株中，每个位点最多鉴定出4个等位基因，在大肠杆菌菌株中最多鉴定出6个等位基因。在这两个物种中，超过一半的AILP位点显示种内多态性。AILP数据应用于李斯特菌和大肠杆菌菌株的系统发育分析。单核细胞增生李斯特菌与李斯特菌属之间有明显区别。此外，该方法将单核细胞增生李斯特菌分为三个已知谱系，并区分出最常见的强毒株血清型组4b。在大肠杆菌中，AILP分析分离出已知组以及强毒O157:H7分离株。李斯特菌和大肠杆菌的这些发现与使用分子标记的其他系统发育研究一致。AILP方法被发现是一种快速、简单、可重复且低成本的初始细菌分型方法，可作为流行病学调查的基础。
