title: Rapid and sensitive detection of lower respiratory tract infections by stuffer-free multiplex ligation-dependent probe amplification.
abstract: Lower respiratory tract infection is one of the most common infectious diseases. However, conventional methods for detecting infectious pathogens are time-consuming, and generally have a limited impact on early therapeutic decisions. We previously reported a rapid and sensitive method for detecting such pathogens using stuffer-free multiplex ligation-dependent probe amplification coupled with high-resolution CE-SSCP. In this study, we report an application of this method to the detection of respiratory pathogens. As originally configured, this method was capable of simultaneously detecting seven bacterial species responsible for lower respiratory tract infections, but its detection limit and assay time were insufficient to provide useful information for early therapeutic decisions. To improve sensitivity and shorten assay time, we added a target-specific preamplification step, improving the detection limit from 50 pg of genomic DNA to 500 fg. We further decreased time requirements by optimizing the hybridization step, enabling the entire assay to be completed within 7 h while maintaining the same detection limit. Taken together, these improvements enable the rapid detection of infectious doses of pathogens (i.e. a few dozen cells), establishing the strong potential of the refined method, particularly for aiding early treatment decisions.
authors: School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea.
authors: Boram
authors: B
authors: Chung
authors: Gi Won
authors: GW
authors: Shin
authors: Chan Kwon
authors: CK
authors: Park
authors: Woong
authors: W
authors: Choi
authors: Yeun-Jun
authors: YJ
authors: Chung
authors: Hyung Kyu
authors: HK
authors: Yoon
authors: Gyoo Yeol
authors: GY
authors: Jung
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics*; D001424:Bacterial Infections / Q000382:microbiology; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D058889:Molecular Typing / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012141:Respiratory Tract Infections / Q000382:microbiology*; D013183:Sputum / Q000382:microbiology
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial
keywords: CE; Multiplex ligation-dependent probe amplification; Pathogen detection; Respiratory tract infection; SSCP