Multiple Cross Displacement Amplification Coupled With Nanoparticles-Based Lateral Flow Biosensor for Detection of Staphylococcus aureus and Identification of Methicillin-Resistant S. aureus

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.

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

“…Thus, modifications of the ddPCR system seem necessary for clinical practice, for example by adding extra channels to the device for additional primer–probe combinations (whether or not on 16S/28S rRNA) to detect pathogen‐specific sequences (e.g. nuc or eap genes for S. aureus species), but also to allow random access when the system is already running (Hussain et al , ; Wang et al , ). Another limitation is that, in line with most molecular tests, the ddPCR set‐up does not provide information regarding microbial susceptibility to antibiotics (Coopersmith et al , ; Pilecky et al , ).…”

<p>Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections</p>

“…The stem-loop structures formed over series of steps from these two cycles of amplification become templates in the exponential amplification cycle and undergo series of amplification and displacement reactions to yield multiple copies of the target DNA bearing inverted repeats. 25 Products of MCDA amplification can be visualised by gel electrophoresis and colourimetric methods 25,155 and also lateral flow biosensor, [155][156][157][158] nanoparticles based biosensor 155,159 and real-time turbidity. 155 Primer design for MCDA uses software used for LAMP and PCR primer design which are PrimerExplorer (Eiken Chemical Company limited, Japan) and Primer Premier (Premier Biosoft International, USA).…”

“…155 MCDA and SAMRS-MCDA have been successfully used to detect disease-causing bacteria such as Shigella species, 158 Vibrio parahaemolyticus, 157 Listeria monocytogenes, 160 Klebsiella pneumonia, 159,161 Pseudomonas aeruginosa, 155 S. aureus and MRSA by the mecA and nuc genes as targets. 156 A multiplex MCDA was also successfully used to distinguish between MRSA, MSSA and non-S. aureus. There have been For personal use only.…”

“…CP2 product displaced by F2 primer from step 2, is extended at the 3ʹ end to form a new double strand with stem loop structure (step 6). Primers C2, D2 and R2 then anneal to the newly formed strands to repeat similar form of amplification by C1, D1 and R1 (step 8 reports of impressive specificity and sensitivity for MCDA in the mentioned studies with detection of target organisms from samples such as sputum, 159,161 blood, 156 oyster, 157 and faeces. 158 With results from the mentioned studies comparable to established methods, MCDA could serve as a valuable onthe-spot diagnostic tool for rapidly detecting disease agents specifically in areas with resource constraints, giving its simplicity and cost-effectiveness.…”

Multiple Cross Displacement Amplification Coupled with Gold Nanoparticles-Based Lateral Flow Biosensor for Detection of the Mobilized Colistin Resistance Gene mcr-1

“…Nevertheless, the requirements of highly sophisticated devices, Strictly experimental environments and well-trained professionals restrict those techniques to apply in resource-challenged areas and “on-site” detection (19). Recently, multiple cross displacement amplification (MCDA), a novel nucleic acid amplification technique, has been utilized in detection of bacterial agents, such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus (MRSA) (20, 21). With the advantages of rapidity, specificity and sensitivity, MCDA operated in a simple heater can yield amplifcons from a few colonies (20, 22), the amplicons are identified by a gold nanoparticles-based lateral flow biosensor (LFB) subsequently.…”