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.
Microb Pathog
; 139: 103862, 2020 Feb.
Article
em En
| MEDLINE
| ID: mdl-31707080
ABSTRACT
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; SKα/SKß/SKγ. Phylogenetic analysis of the major variable region of SKß (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 Tsp45Iâ³ 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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Filogenia
/
Streptococcus
/
Estreptoquinase
/
Polimorfismo de Fragmento de Restrição
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Reação em Cadeia da Polimerase
/
Alelos
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article