Abstract
Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), is one of the most important human pathogens, which is responsible for bacteremia, soft-tissue infections, and food poisoning. Hence, multiple cross displacement amplification (MCDA) is employed to detect all S. aureus strains, and differentiates MRSA from methicillin-sensitive S. aureus. Multiplex MCDA (m-MCDA), which targets the nuc gene (S. aureus-specific gene) and mecA gene (encoding penicillin-binding protein-2′), could detect S. aureus strains and identify MRSA within 85 min. Detection of the m-MCDA products is achieved using disposable lateral flow biosensors. A total of 58 strains, including various species of Gram-positive and Gram-negative strains, are used for evaluating and optimizing m-MCDA assays. The optimal amplification condition is found to be 63°C for 40 min, with detection limits at 100 fg DNA/reaction for nuc and mecA genes in the pure cultures, and 10 CFU/tube for nuc and mecA genes in the blood samples. The analytical specificity of m-MCDA assay is of 100%, and no cross-reactions to non-S. aureus strains are produced according to the specificity testing. Particularly, two additional components, including AUDG enzyme and dUTP, are added into the m-MCDA amplification mixtures, which are used for eliminating the unwanted results arising from carryover contamination. Thus, the m-MCDA technique appears to be a simple, rapid, sensitive, and reliable assay to detect all S. aureus strains, and identify MRSA infection for appropriate antibiotic therapy.
Highlights
Staphylococcus aureus (S. aureus) is a commensal organism, and approximately of 30% of human population is colonized with the bacterium (Tong et al, 2015)
Advanced assays are needed for rapid detection and accurate differentiation of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) infections to ensure optimal therapy and management of patients
As a potentially valuable tool for the rapid diagnosis of pathogen infection, we report on a method for detection of MSSA and MRSA by multiple cross displacement amplification (MCDA)-Lateral Flow Biosensor (LFB) assay and attempt to investigate the potential clinical impact of the more rapid provision of examination results
Summary
Staphylococcus aureus (S. aureus) is a commensal organism, and approximately of 30% of human population is colonized with the bacterium (Tong et al, 2015). The pathogen is one of the common causes of food-borne disease, and is one of the most important bacteria in hospitalacquired and community-acquired infections related to high mortality (Stryjewski and Corey, 2014). Since the introduction of methicillin, the methicillin-resistant S. aureus (MRSA) has occurred and spread worldwide, and the infections caused by MRSA have become a serious problem and were associated with significant mortality and morbidity, especially in patients with bacteremia (Jacob and DiazGranados, 2013). The MRSA has emerged in over 60% of isolated S. aureus, and the occurrence of methicillin resistance in S. aureus causing infection has exceeded 49% in the United States hospitals and increased steadily (Jernigan, 2004; Misawa et al, 2007). Advanced assays are needed for rapid detection and accurate differentiation of methicillin-susceptible S. aureus (MSSA) and MRSA infections to ensure optimal therapy and management of patients
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