Żaczek Anna, Brzostek Anna, Wojtasik Arkadiusz, Dziadek Jarosław, Sajduda Anna
Department of Biochemistry and Cell Biology, Faculty of Biology and Agriculture, University of Rzeszów, Ćwiklińskiej 2, 35-601 Rzeszów, Poland.
Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106, 93-232 Łódź, Poland.
Biomed Res Int. 2013;2013:865197. doi: 10.1155/2013/865197. Epub 2013 Dec 17.
In this study, 155 clinical Mycobacterium tuberculosis isolates were subject to genotyping with fast ligation-mediated PCR (FLiP). This typing method is a modified mixed-linker PCR, a rapid approach based on the PCR amplification of HhaI restriction fragments of genomic DNA containing the 3' end of IS6110 and resolving the amplicons by polyacrylamide gel electrophoresis. The results were compared with previous data of the more commonly used methods, 15-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and, to verify combined FLiP/MIRU-VNTR clusters, the reference IS6110 restriction fragment length polymorphism (RFLP). FLiP banding patterns were highly reproducible and polymorphic. This method differentiated 119 types among the study set compared to 108 distinct MIRU-VNTR profiles. The discriminatory power of FLiP was slightly higher than that of MIRU-VNTR analysis (Hunter-Gaston Discriminatory Index = 0.991 and 0.990, resp.). Detailed comparison of the clusters defined by each of the methods revealed, however, a more apparent difference in the discriminatory abilities that favored FLiP. Clustering of strains by using combined results of these two PCR-based methods correlated well with IS6110 RFLP-defined clusters, further confirming high discriminatory potential of FLiP typing. These results indicate that FLiP could be an attractive and valuable secondary typing technique for verification of MIRU-VNTR clusters of M. tuberculosis strains.
在本研究中,对155株临床结核分枝杆菌分离株进行了快速连接介导PCR(FLiP)基因分型。这种分型方法是一种改良的混合连接子PCR,是一种基于对含有IS6110 3'端的基因组DNA的HhaI限制性片段进行PCR扩增并通过聚丙烯酰胺凝胶电泳解析扩增子的快速方法。将结果与更常用方法(15位点分枝杆菌散布重复单位-可变数目串联重复序列(MIRU-VNTR)分型)的先前数据进行比较,并为验证FLiP/MIRU-VNTR联合聚类,采用参考IS6110限制性片段长度多态性(RFLP)方法。FLiP条带模式具有高度的可重复性和多态性。与108种不同的MIRU-VNTR图谱相比,该方法在研究集中区分出119种类型。FLiP的鉴别力略高于MIRU-VNTR分析(Hunter-Gaston鉴别指数分别为0.991和0.990)。然而,对每种方法所定义聚类的详细比较显示,鉴别能力上存在更明显的差异,FLiP更具优势。通过这两种基于PCR的方法的联合结果对菌株进行聚类与IS6110 RFLP所定义的聚类相关性良好,进一步证实了FLiP分型具有较高的鉴别潜力。这些结果表明,FLiP可能是一种有吸引力且有价值的辅助分型技术,用于验证结核分枝杆菌菌株的MIRU-VNTR聚类。