Multiplex identification of drug-resistant Gram-positive pathogens using stuffer-free MLPA system

作者：Chung Boram; Park Chulmin; Cho Sung Yeon; Shin Sun; Yim Seon Hee; Jung Gyoo Yeol; Lee Dong Gun; Chung Yeun Jun^*

来源：Electrophoresis, 2016, 37(23-24): 3079-3083.

DOI：10.1002/elps.201600372

• Capillary electrophoresis
• Gram-positive pathogen
• Multiplex ligation-dependent probe amplification
• Sepsis

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.

• 出版日期2016-12