de la Puente-Redondo V A, del Blanco N G, Gutiérrez-Martín C B, García-Peña F J, Rodríguez Ferri E F
Section of Microbiology and Immunology, Department of Animal Health, Faculty of Veterinary Medicine, León, Spain.
J Clin Microbiol. 2000 Mar;38(3):1016-22. doi: 10.1128/JCM.38.3.1016-1022.2000.
In this study, we evaluated three PCR methods for epidemiological typing of Francisella tularensis: repetitive extragenic palindromic element PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and random amplified polymorphic DNA (RAPD) assay with both M13 and T3-T7 primers. The analysis was performed with 40 strains of F. tularensis isolated from hares, humans, ticks, and a vole. On the basis of the combination of REP, ERIC, and RAPD fingerprints, F. tularensis strains were divided into 17 genetic groups (designated A to Q), and one Francisella novicida strain was classified in group R. The F. novicida strain is of special concern, since previous genetic methods have been unable to clearly distinguish between F. tularensis and F. novicida. The F. tularensis isolates recovered from hares were included in groups A to J, M, and P; those recovered from humans were included in groups A, D, G, J, L, O, and N; those isolated from ticks were included in groups B and Q; and that recovered from a vole was in group K. The diversities calculated for the 40 F. tularensis isolates, according to Simpson's index, were 0.14 for REP-PCR, 0.52 for ERIC-PCR, 0.39 for RAPD assay with the M13 primer (RAPD/M13-PCR), and 0.65 for RAPD/T3-T7-PCR, and the diversity increased up to 0.90 when ERIC-PCR, RAPD/M13-PCR, and RAPD/T3-T7-PCR were combined. Our results suggest that although limited genetic heterogeneity among F. tularensis strains was observed, this small variation is enough to validate the PCR methods used in this study and their combinations, because they can provide safe, useful, and rapid tools for the typing of F. tularensis.
在本研究中，我们评估了三种用于土拉弗朗西斯菌流行病学分型的聚合酶链反应（PCR）方法：重复外显子回文元件PCR（REP-PCR）、肠杆菌重复基因间共有序列PCR（ERIC-PCR）以及使用M13和T3-T7引物的随机扩增多态性DNA（RAPD）分析。对从野兔、人类、蜱虫和一只田鼠中分离出的40株土拉弗朗西斯菌进行了分析。根据REP、ERIC和RAPD指纹图谱的组合，土拉弗朗西斯菌菌株被分为17个基因群（命名为A至Q），一株新凶手弗朗西斯菌菌株被归类为R群。新凶手弗朗西斯菌菌株值得特别关注，因为之前的基因方法无法清晰区分土拉弗朗西斯菌和新凶手弗朗西斯菌。从野兔中分离出的土拉弗朗西斯菌分离株包括在A至J、M和P群；从人类中分离出的包括在A、D、G、J、L、O和N群；从蜱虫中分离出的包括在B和Q群；从田鼠中分离出的在K群。根据辛普森指数计算的40株土拉弗朗西斯菌分离株的多样性，REP-PCR为0.14，ERIC-PCR为0.52，使用M13引物的RAPD分析（RAPD/M13-PCR）为0.39，RAPD/T3-T7-PCR为0.65，当ERIC-PCR、RAPD/M13-PCR和RAPD/T3-T7-PCR组合时，多样性增加至0.90。我们的结果表明，尽管观察到土拉弗朗西斯菌菌株之间的遗传异质性有限，但这种微小变异足以验证本研究中使用的PCR方法及其组合，因为它们可为土拉弗朗西斯菌的分型提供安全、有用且快速的工具。
