title: A locked nucleic acid (LNA)-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.
abstract: Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.
authors.affiliation: CAS Key Laboratory of Genome Sciences & Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China; Department of Immunology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
authors.forename: Lingxiang
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Department of Clinical Microbiology, General Hospital of People's Liberation Army, Beijing, China.
authors.forename: Dingxia
authors.initials: D
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Qiming
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: Department of Immunology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
authors.forename: Zexia
authors.initials: Z
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Genome Sciences & Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Fang
authors.affiliation: Department of Immunology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
authors.forename: Quan-Zhen
authors.initials: QZ
authors.lastname: Li
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016470:Bacteremia; D001419:Bacteria / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D001424:Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests; D009841:Oligonucleotides; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000900:Anti-Bacterial Agents; D009841:Oligonucleotides; C477371:locked nucleic acid