Rational design of DNA sequence-based strategies for subtyping Listeria monocytogenes
Rational design of DNA sequence-based strategies for subtyping Listeria monocytogenes
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DOI：
10.1128/jcm.40.9.3319-3325.2002
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发表时间：
2002-09-01
影响因子：
9.4
通讯作者：
Wiedmann, M
中科院分区：
文献类型：
--
作者：
Cai, S;Kabuki, DY;Wiedmann, M
区分细菌的能力超越了物种水平，对于识别和跟踪传染病爆发以及提高我们对细菌病原体的群体遗传学，流行病学和生态学的认识至关重要。常用的亚型分型方法，如血清分型、噬菌体分型、核糖体分型和脉冲场凝胶电泳，可能产生模糊的结果，难以标准化和在实验室之间共享。基于DNA序列的分型策略可以减少解释歧义。我们报告了一个合理的方法设计基于序列的分型方法的发展。选择单核细胞增生李斯特菌作为模式生物来测试这种方法的有效性。测定了15株L的2个管家基因（recA和inlA）、1个胁迫反应基因（sigB）、2个毒力基因（actA和inlA）和2个基因间区（hly-mpl和plcA-hly）的序列。单核细胞增生分离株。从超过1，000 L的代表性采集物中选择分离株。单核细胞增生分离株，以反映该物种的遗传多样性。比对DNA序列，并对每个基因进行滑动窗口分析，以确定600 bp长的区域，这些区域是（i）最多态的（使用ProSeq）或（ii）最具区分性的（使用WINDOWMIN中实现的新算法）。actA（1，929 bp）和inlA（2，235 bp）的完整基因序列提供了最高的区分度（分别鉴定15和14个等位基因类型）。WINDOWMIN允许在这些基因内鉴定600-bp区域，其提供类似的区分能力（分别产生15和13个等位基因类型）。管家和应激反应基因中最具歧视性的600 bp片段将分离株分为8至10个亚型;基因间区域序列分别基于hly-mpl和plcA-hly的335和242 bp序列产生8和12个等位基因类型。被鉴定为最多态的区域不一定是最具区分性的;因此，应用WINDOWMIN算法为确定基于DNA序列的亚型的最佳靶区域提供了强大的工具。我们的具体结果还表明，包括毒力基因靶序列的DNA序列为基础的亚型计划，为L。单核细胞增多症是实现最大亚型分化所必需的。
The ability to differentiate bacteria beyond the species level is essential for identifying and tracking infectious disease outbreaks and to improve our knowledge of the population genetics, epidemiology, and ecology of bacterial pathogens. Commonly used subtyping methods, such as serotyping, phage typing, ribotyping, and pulsed-field gel electrophoresis, can yield ambiguous results that are difficult to standardize and share among laboratories. DNA sequence-based subtyping strategies can reduce interpretation ambiguity. We report the development of a rational approach for designing sequence-based subtyping methods. Listeria monocytogenes was selected as the model organism for testing the efficacy of this approach. Two housekeeping genes (recA and prs), one stress response gene (sigB), two virulence genes (actA and inlA), and two intergenic regions (hly-mpl and plcA-hly) were sequenced for 15 L. monocytogenes isolates. Isolates were chosen from a representative collection of more than 1,000 L. monocytogenes isolates to reflect the genetic diversity of this species. DNA sequences were aligned, and sliding window analyses were performed for each gene to define 600-bp-long regions that were (i) most polymorphic (using ProSeq) or (ii) most discriminatory (using a new algorithm implemented in WINDOWMIN). Complete gene sequences for actA (1,929 bp) and inlA (2,235 bp) provided the highest discrimination (identifying 15 and 14 allelic types, respectively). WINDOWMIN allowed identification of 600-bp regions within these genes that provided similar discriminatory power (yielding 15 and 13 allelic types, respectively). The most discriminatory 600-bp fragments identified in the housekeeping and stress response genes differentiated the isolates into 8 to 10 subtypes; intergenic region sequences yielded 8 and 12 allelic types based on 335- and 242-bp sequences for hly-mpl and plcA-hly, respectively. Regions identified as most polymorphic were not necessarily most discriminatory; therefore, application of the WINDOWMIN algorithm provided a powerful too[ for determining the best target regions for DNA sequence-based subtyping. Our specific results also show that inclusion of virulence gene target sequences in a DNA sequence-based subtyping scheme for L. monocytogenes is necessary to achieve maximum subtype differentiation.