Abstract
BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).ResultsThe protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.ConclusionsThe established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.
Highlights
Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world
Despite none of significant difference observed, the electrophoresis of loop-mediated isothermal amplification (LAMP) products which were amplified under 64°C exhibited slightly larger amount of DNA amplicons when compared to other temperatures (Figure 1), which was consistent with studies previously
The first study on detection of MRSA by LAMP assay was reported lately [6], 3 individual LAMP assays with targets including 16SrRNA, femA and mecA were laborious and time and expense demanding
Summary
Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA). Since the first report in 1961, methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world, which has raised a global challenge for clinicians, hospital epidemiologists and administrators [10,11,12,13,14,15,16]. The mecA gene, encoding the PBP2a protein and causing methicillin resistance in staphylococci, is located on staphylococcal cassette chromosome mec (SCCmec). The increasing awareness for the risk and hazard of MRSA strains raised the demands of an early, cost-effective, timely, specific and sensitive detection assay [5,6,10,20]
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