Multiplex identification of drug-resistant Gram-positive pathogens using stuffer-free MLPA system.
Electrophoresis
; 37(23-24): 3079-3083, 2016 12.
Article
em En
| MEDLINE
| ID: mdl-27573990
ABSTRACT
Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture-based methods require relatively longer time to identify drug-resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug-resistant Gram-positive pathogens, we developed a method by using stuffer-free multiplex ligation-dependent probe amplification (MLPA) coupled with high-resolution CE single-strand conformation polymorphisms (CE-SSCP) system. We designed three probe sets for genes specific to Gram-positive species (Staphylococcus aureus nuc, Enterococcus faecium sodA, and Streptococcus pneumoniae lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram-positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain-specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target-specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug-resistant Gram-positive pathogens from sepsis patients' blood.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Infecções por Bactérias Gram-Positivas
/
Eletroforese Capilar
/
Reação em Cadeia da Polimerase Multiplex
/
Bactérias Gram-Positivas
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Limite:
Humans
Idioma:
En
Revista:
Electrophoresis
Ano de publicação:
2016
Tipo de documento:
Article