[Establishment and evaluation of a rapid nucleic acid detection method for methicillin-resistant Staphylococcus...

To establish a rapid and accurate nucleic acid-based point-of-care detection assay for methicillin-resistant Staphylococcus aureus (MRSA). We designed specific primers and fluorescent probes targeting 16S rRNA, nuc, and mecA genes. Gradient DNA (deoxyribonucleic acid) standards prepared from an MRSA reference strain were used to optimize the qPCR system, and amplicons and T-A cloning plasmids were sequenced for verification. Subsequently, the established multiplex real-time PCR assay was systematically evaluated for sensitivity, linearity, precision, specificity, and interference tolerance. Finally, the optimized method was applied to clinical samples, with mass spectrometry-a commonly used clinical laboratory identification method-serving as the reference standard to validate its practical value. The assay completed detection within 30 min, with limits of detection of 1.0×10², 1.0×10², and 1.0×10³ CFU/mL and linear ranges of 1.0×10²‒1.0×10⁹, 1.0×10²‒1.0×10⁹, and 1.0×10³‒1.0×10⁹ CFU/mL for 16S rRNA, nuc, and mecA, respectively. Intra- and inter-batch coefficients of variation were <5%, and no cross-reactivity was observed. Neither hemoglobin (2 g/L) nor 100 μg/mL ceftriaxone sodium interfered with the detection results. The method showed high concordance, sensitivity, and specificity versus MALDI-TOF MS. In conclusion, the MRSA nucleic-acid detection assay developed in this study combines high sensitivity with operational simplicity, providing a new strategy for point-of-care pathogen nucleic-acid testing and serving as a reliable tool for rapid clinical microbial screening.