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首页> 外文期刊>Microbial Pathogenesis >In silico design and in vitro validation of a novel PCR-RFLP assay for determination of phylogenetic clusters of streptokinase gene alleles in streptococci groups
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In silico design and in vitro validation of a novel PCR-RFLP assay for determination of phylogenetic clusters of streptokinase gene alleles in streptococci groups

机译：在硅设计和新型PCR-RFLP测定的体外验证中，用于测定链球菌组中的链孢菌素基因等内燃机的系统发育簇

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摘要

Streptokinase (SK), a heterogeneous plasminogen (Pg) activator protein secreted by groups A, C and G streptococci (GAS/GCS/GGS) is a virulence factor composed of three structural domains; SK alpha/SK beta/SK gamma. Phylogenetic analysis of the major variable region of SK beta (sk-V1; nucleotides 448-791; 343bp) which classifies the SK alleles into SK1/SK2 clusters and SK2a/SK2b sub-clusters, is an approved assay to categorize clinical/natural streptococcal-isolates into co-related functional/pathogenesis groups. Herein, we describe a novel PCR-RFLP assay that in combination with Numerical Taxonomy and multivariate analysis System (NTSYS) resulted to dendrograms with complete adaption to that of the phylogenetic analysis of sk-V1-based clustering. In silico analyses by 30 restriction enzymes on GenBank-acquired sk-V1 sequences of known streptococcal clusters, resulted to the selection of "BsrI, MseI and Tsp45I" enzymes that produced proper patterns to construct the expected dendrograms. In vitro analysis of the selected enzymes on clinical isolates of GAS/GCS/GGS validated the production of the same in silico-observed digestion patterns. Comparison of the constructed dendrogram and phylogenetic trees of selected GenBank and clinical isolates of streptococci indicated complete adaptation. Assessment of Pg-activation activity in selected clinical isolates indicated the expected co-related functionalities of the classified SK-clusters by the invented PCR-RFLP/NTSYS method. The simplicity of the assay relieves the need of sequencing/phylogenetic analyses for SK-clustering.
机译：链孢菌素（SK），由A，C和G链球菌（气体/ GCS / GG）分泌的异质纤溶酶原（PG）活化剂蛋白是由三个结构域组成的毒力因子; sk alpha / sk beta / sk gamma。 SKβ（SK-V1;核苷酸448-791; 343bp）的主要可变区的系统发育分析，其将SK等位基因分类为SK1 / SK2簇和SK2A / SK2B子簇，是批准的测定，以分类临床/天然链球菌 - 分为与共同相关的功能/发病机制组。在此，我们描述了一种新的PCR-RFLP测定，其与数值分类和多变量分析系统（NTSYS）组合导致树枝状图，其具有完全适应SK-V1基团聚类的系统发育分析。在硅藻中，通过30次限制酶对已知的链球菌簇的Genbank获得的SK-V1序列，导致选择“BSRI，MSEI和TSP45i”的酶，这些酶产生适当的图案以构建预期的树状图。对临床分离株的选定酶的体外分析验证了硅观察到的消化模式中的相同的产生。所选择的成因和临床分离株构建的树状图和系统发育树的比较表明了完全适应。选择临床分离株中PG活化活性的评估表明了本发明的PCR-RFLP / NTSYS方法的分类SK-簇的预期共同相关官能。测定的简单性降低了需要对SK聚类进行测序/系统发育分析。

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