title: Development of a nucleic acid lateral flow immunoassay (NALFIA) for reliable, simple and rapid detection of the methicillin resistance genes mecA and mecC.
abstract: The gene mecA and its homologue mecC confer methicillin resistance in Staphylococcus aureus and other staphylococci. Methicillin-resistant staphylococci (MRS) are considered resistant to all β-lactam antibiotics. To avoid the use of β-lactam antibiotics for the control of MRS infections, there is an urgent need for a fast and reliable screening assay for mecA and mecC that can easily be integrated in routine laboratory diagnostics. The aim of this study was the development of such a rapid detection method for methicillin resistance based on nucleic acid lateral flow immunoassay (NALFIA) technology. In NALFIA, the target sequences are PCR-amplified, immobilized via antigen-antibody interaction and finally visualized as distinct black bars resulting from neutravidin-labeled carbon particles via biotin-neutravidin interaction. A screening of 60 defined strains (MRS and non-target bacteria) and 28 methicillin-resistant S. aureus (MRSA) isolates from clinical samples was performed with PCR-NALFIA in comparison to PCR with subsequent gel electrophoresis (PCR-GE) and real-time PCR. While all samples were correctly identified with all assays, PCR-NALFIA was superior with respect to limits of detection. Moreover, this assay allowed for differentiation between mecA and mecC by visualizing the two alleles at different positions on NALFIA test stripes. However, since this test system only targets the mecA and mecC genes, it does not allow to determine in which staphylococcal species the mec gene is included. Requiring only a fraction of the time needed for cultural methods (i.e. the gold standard), the PCR-NALFIA presented here is easy to handle and can be readily integrated into laboratory diagnostics.
authors: Q-Bioanalytic GmbH, Bremerhaven, Germany; Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany.
authors: Constanze
authors: C
authors: Seidel
authors: Q-Bioanalytic GmbH, Bremerhaven, Germany.
authors: Sonja
authors: S
authors: Peters
authors: Q-Bioanalytic GmbH, Bremerhaven, Germany.
authors: Erik
authors: E
authors: Eschbach
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany.
authors: Andrea T
authors: AT
authors: Feßler
authors: Q-Bioanalytic GmbH, Bremerhaven, Germany. Electronic address: oberheitmann@q-bioanalytic.com.
authors: Boris
authors: B
authors: Oberheitmann
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany. Electronic address: stefan.schwarz@fli.de.
authors: Stefan
authors: S
authors: Schwarz
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D007118:Immunoassay / Q000379:methods / Q000662:veterinary*; D016106:Methicillin Resistance / Q000235:genetics; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification; D046915:Penicillin-Binding Proteins / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D013210:Staphylococcus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams; C487789:mecA protein, Staphylococcus aureus
keywords: Antibiotic resistance; Methicillin resistant staphylococci; PCR-NALFIA; Real-time PCR; Staphylococcus aureus