The aim of this study was to develop a rapid detection assay to identify methicillin-resistant Staphylococcus aureus by simultaneous testing for the mecA, nuc, and femB genes using the loop-mediated isothermal amplification (LAMP) method. LAMP primers were designed using online bio-software (http://primerexplorer.jp/e/), and amplification reactions were performed in an isothermal temperature bath. The products were then examined using 2% agarose gel electrophoresis. MecA, nuc, and femB were confirmed by triplex TaqMan real-time PCR. For better naked-eye inspection of the reaction result, hydroxy naphthol blue (HNB) was added to the amplification system. Within 60 min, LAMP successfully amplified the genes of interest under isothermal conditions at 63 °C. The results of 2% gel electrophoresis indicated that when the Mg2+ concentration in the reaction system was 6 μmol, the amplification of the mecA gene was relatively good, while the amplification of the nuc and femB genes was better at an Mg2+ concentration of 8 μmol. Obvious color differences were observed by adding 1 μL (3.75 mM) of HNB into 25 μL reaction system. The LAMP assay was applied to 128 isolates cases of methicillin-resistant Staphylococcus aureus, which were separated from the daily specimens and identified by Vitek microbial identification instruments. The results were identical for both LAMP and PCR. LAMP offers an alternative detection assay for mecA, nuc, and femB and is faster than other methods.
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This work was supported by the National Natural Science Foundation (No. 81401311) and the Capital Characteristic Clinical Application Research (WU JIEPING Foundation No. Z141107006614009). This work was also supported by the Navy General Hospital Innovation Cultivation Foundation (No. CXPY201412).
CQY and ZQY conceived the study, collected and analyzed the data, and drafted the manuscript. GJW and LYJ conceived the project and provided technical support for data collection and analysis. CCG conceived the study and revised the manuscript. All authors read and approved the final manuscript.
Compliance with Ethical Standards
Conflict of interest
The authors declared that they have no competing interests.
The subjects of this work are bacteria that were separated and purified from clinical samples. This article does not contain any studies with human or animals performed by any of the authors.
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