Rapid and simple detection of methicillin-resistance Staphylococcus aureus by orfX loop-mediated isothermal amplification assay

Abstract

Background: Methicillin-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).

Results: The 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.

Conclusions: The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.

Figure 1

Electrophoresis of orfX-LAMP. A: Under different temperatures. B: Detection limit, lane 1-8: negative control (distilled water), DNA Marker, negative contro1 (template DNA sample of other bacteria), 1 copies, 10 copies, 10² copies, 10³ copies and 10⁴ copies.

Figure 2

Evaluation of orfX-LAMP assay under different time points. A: Electrophoresis of orfX-LAMP reaction under different time points: 1-10: DNA Marker, 15, 30, 45, 60, 75, 90, 105 and 120 min, negative control. B: Sybr Green of orfX-LAMP reaction under different time points.

Figure 3

Result determination of orfX-LAMP assays by color change. A: Results determination of orfX-LAMP by naked eyes. B: Results determination of orfX-LAMP by Sybr Green with dark background, light backgrounds and under UV light. C: Results determination of orfX-LAMP in application by Sybr Green.