Rapid detection of methicillin-resistant Staphylococcus aureus in positive blood-cultures by recombinase polymerase amplification combined with lateral flow strip

• Published in: PloS one 2022-06, Vol.17 (6), p.e0270686
• Main Authors: Srisrattakarn, Arpasiri, Panpru, Pimchanok, Tippayawat, Patcharaporn, Chanawong, Aroonwadee, Tavichakorntrakool, Ratree, Daduang, Jureerut, Wonglakorn, Lumyai, Lulitanond, Aroonlug
• Format: Article
• Language: English
• Subjects: Age; Amplification; Analysis; Antibiotic resistance; Biology and life sciences; Blood; Care and treatment; Deoxyribonucleic acid; Design; DNA; DNA probes; Drug resistance; Drug resistance in microorganisms; Genes; Health aspects; MecA protein; Medicine and health sciences; Methicillin; Methicillin-Resistant Staphylococcus aureus - genetics; Methods; Mortality; Nucleic Acid Amplification Techniques - methods; Nucleotidyltransferases; Pathogens; Penicillin; Penicillin-binding protein; Penicillin-binding protein 2a; Protein binding; Proteins; Public health; Recombinase; Recombinases - genetics; Research and Analysis Methods; Room temperature; Sensitivity; Sensitivity and Specificity; Staphylococcus aureus; Staphylococcus aureus - genetics; Staphylococcus infections; Strip; Thermonuclease
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0270686

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is an important bacterium that causes community and healthcare-related infections throughout the world. However, the current conventional detection methods are time-consuming. We therefore developed and evaluated a recombinase polymerase amplification-lateral flow strip (RPA-LF) approach for detection of MRSA in positive blood-culture samples. Sixty positive blood-cultures from a hospital were tested directly without DNA extraction and purification before the amplification reaction. RPA primers and probes were designed for nuc (encoding thermonuclease) and mecA (encoding penicillin-binding protein 2a) genes to diagnose S. aureus and its methicillin-resistance status. The RPA reaction occurred under isothermal conditions (45°C) within 20 min and a result was provided by the LF strip in a further 5 min at room temperature. The evaluation of RPA-LF using blood-culture samples showed 93.3% (14/15) sensitivity for identifying S. aureus, and no cross-amplification was seen [100% (45/45) specificity]. For detection of methicillin resistance, the RPA-LF test provided 100% (16/16) sensitivity and 97.7% (43/44) specificity. The RPA-LF is rapid, highly sensitive, robust and easy to use. It can be used for direct detection of MRSA with no requirement for special equipment.