title: Multiplex identification of drug-resistant Gram-positive pathogens using stuffer-free MLPA system.
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.
authors: Department of Microbiology, College of Medicine, The Catholic University of Korea Korea, Seoul, Korea.|Integrated Research Center for Genome Polymorphism, College of Medicine, The Catholic University of Korea Korea, Seoul, Korea.
authors: Boram
authors: B
authors: Chung
authors: Vaccine Bio Research Institute, College of Medicine, The Catholic University of Korea Korea, Seoul, Korea.
authors: Chulmin
authors: C
authors: Park
authors: Vaccine Bio Research Institute, College of Medicine, The Catholic University of Korea Korea, Seoul, Korea.|Division of Infectious Diseases, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea.
authors: Sung-Yeon
authors: SY
authors: Cho
authors: Department of Microbiology, College of Medicine, The Catholic University of Korea Korea, Seoul, Korea.|Integrated Research Center for Genome Polymorphism, College of Medicine, The Catholic University of Korea Korea, Seoul, Korea.
authors: Sun
authors: S
authors: Shin
authors: Integrated Research Center for Genome Polymorphism, College of Medicine, The Catholic University of Korea Korea, Seoul, Korea.
authors: Seon-Hee
authors: SH
authors: Yim
authors: School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.
authors: Gyoo Yeol
authors: GY
authors: Jung
authors: Vaccine Bio Research Institute, College of Medicine, The Catholic University of Korea Korea, Seoul, Korea.|Division of Infectious Diseases, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea.
authors: Dong-Gun
authors: DG
authors: Lee
authors: Department of Microbiology, College of Medicine, The Catholic University of Korea Korea, Seoul, Korea.|Integrated Research Center for Genome Polymorphism, College of Medicine, The Catholic University of Korea Korea, Seoul, Korea.
authors: Yeun-Jun
authors: YJ
authors: Chung
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D006094:Gram-Positive Bacteria / Q000235:genetics*; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology*; D006801:Humans; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D018805:Sepsis / Q000382:microbiology
keywords: Capillary electrophoresis; Gram-positive pathogen; Multiplex ligation-dependent probe amplification; Sepsis