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首页> 外文期刊>Journal of Clinical Microbiology >Evaluation of PCR-Based Methods for Discrimination ofFrancisella Species and Subspecies and Development of a Specific PCR That Distinguishes the Two Major Subspecies of Francisella tularensis
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Evaluation of PCR-Based Methods for Discrimination ofFrancisella Species and Subspecies and Development of a Specific PCR That Distinguishes the Two Major Subspecies of Francisella tularensis

机译：基于PCR的弗朗西斯菌种和亚种的鉴别方法的评估，以及可区分图拉弗朗西斯菌两个主要亚种的特异性PCR的发展

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Previous studies have demonstrated that the four subspecies of the human pathogen Francisella tularensis, despite showing marked variations in their virulence for mammals and originating from different regions in the Northern Hemisphere, display a very close phylogenetic relationship. This property has hampered the development of generally applicable typing methods. To overcome this problem, we evaluated the use of PCR for discrimination of the subspecies using various forms of long arbitrary primers or primers specific for repetitive extragenic palindromic sequences (REP) or enterobacterial repetitive intragenic consensus (ERIC) sequences. Patterns generated by use of REP, ERIC, or long arbitrary primers allowed differentiation at the species level and of the four subspecies of F. tularensis. With each of these three methods, similar or identical clustering of strains was found, and groups of strains of different geographical origins or differing in virulence showed distinct patterns. The discriminatory indices of the methods varied from 0.57 to 0.65; thus, the patterns were not sufficiently discriminatory to distinguish individual strains. The sequence of a fragment generated by amplification with an arbitrary primer was determined, and a region showing interstrain heterogeneity was identified. Specific primers were designed, and a PCR was developed that distinguished strains of F. tularensis subsp.holarctica from strains of other F. tularensissubspecies, including strains of the highly virulent F. tularensis subsp. tularensis. Notably, one European isolate showed the genetic pattern typical of the highly virulentF. tularensis subsp. tularensis, generally believed to exist only in North America. It is proposed that a combination of the specific PCR together with one method generating subspecies-specific patterns is suitable as a rapid and relatively simple strategy for discrimination of Francisella species and subspecies.
机译：先前的研究表明，人类病原体 Francisella tularensis 的四个亚种尽管显示出它们对哺乳动物的毒力显着变化并且起源于北半球的不同区域，但它们之间的亲缘关系却非常密切。此属性阻碍了通常适用的键入方法的开发。为了克服这个问题，我们评估了使用PCR鉴别亚种的方法，即使用各种形式的长任意引物或重复性外基因回文序列（REP）或肠细菌性重复基因内共有序列（ERIC）特异性的引物。通过使用REP，ERIC或任意长引物生成的模式允许在种水平和 F的四个亚种之间进行区分。 tularensis 。使用这三种方法中的每一种，都发现了相似或相同的菌株簇，并且不同地理起源或不同毒力的菌株组显示出不同的模式。该方法的鉴别指数从0.57到0.65不等。因此，这些模式不足以区分各个菌株。确定通过用任意引物扩增产生的片段的序列，并鉴定出显示菌株间异质性的区域。设计了特异性引物，并开发了可区分 F菌株的PCR。来自其他 F菌株的土拉李亚种 holarctica 。 tularensis 亚种，包括高毒力 F菌株。 tularensis 子亚种 tularensis 。值得注意的是，一种欧洲分离株显示出高毒性 F的典型遗传模式。 tularensis 子亚种 tularensis ，通常被认为仅存在于北美。建议将特异性PCR与一种产生亚种特异性模式的方法相结合，适合作为一种快速，相对简单的策略来区分弗朗西斯菌种和亚种。

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