The rapid and visual detection of methicillin-susceptible and methicillin-resistant Staphylococcus aureus using multiplex loop-mediated isothermal amplification linked to a nanoparticle-based lateral flow biosensor

Abstract

Background Staphylococcus aureus ( S. aureus ), including methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA), is an eminent human pathogen that can colonize the human host and cause severe life-threatening infections. The development of a reliable, simple and rapid assay for detecting S. aureus and identifying MRSA is important for diagnosis and follow-up treatment. Methods A novel molecular diagnosis technique, named multiplex loop-mediated isothermal amplification linked to a nanoparticle-based lateral flow biosensor (m-LAMP-LFB), was applied to detect all S. aureus species and identify MRSA. Two sets of primers were designed based on the femA gene ( S. aureus -specific gene) and the mecA gene (encoding penicillin-binding protein 2a), and the multiple-LAMP products were analyzed using LFB. The m-LAMP-LFB amplification conditions, including the target DNA concentration, reaction temperature and time, were optimized. The sensitivity and specificity of the m-LAMP-LFB method were tested in the current study, and the multiple-LAMP-LFB technology was applied to detect the MSSA and MRSA strains from clinical samples. Results The S. aureus- and MRSA-specific primers based on the femA and mecA genes allowed the multiple-LAMP technology to detect S. aureus and MRSA, respectively. The multiple-LAMP conditions were optimized at 63 °C for 40 min. The full process, including genomic DNA template preparation, LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the multiple-LAMP assay for femA and mecA detection was 100 fg of genomic DNA template per reaction. The specificity of m-LAMP-LFB detection was 100 %, and no cross-reactions to non- S. aureus strains were observed. Conclusion The multiple-LAMP-LFB technique developed in the current study is a reliable, simple, rapid, specific and sensitive method to identify MSSA and MRSA infections for appropriate antibiotic therapy.

A New Optical Fiber Probe-Based Quantum Dots Immunofluorescence Biosensors in the Detection of Staphylococcus aureus

“…For the specificity, we adopt a double-antibody sandwich structure to capture the S. aureus and the high specificity could be guaranteed based on the principle of antigen-antibody specific binding. While the various ultra-sensitive biosensors (Li et al, 2018;Andersson et al, 2020;Chen et al, 2020) based on the molecular detection technology of S. aureus-related genes may lead to excessively high false positive rate. In particular, even a small amount of contamination in clinical samples can lead to false positives.…”

Recent Developments in Phenotypic and Molecular Diagnostic Methods for Antimicrobial Resistance Detection in Staphylococcus aureus: A Narrative Review

“…The author tested this method in 63 clinical whole blood samples from patients suspected of being infected with S. aureus . The biosensor correctly detected 12 MRSA, 16 MSSA and 35 S. aureus -negative isolates, reaching a sensitivity and specificity of 100% compared to cultures [ 125 ]. Several other molecular biosensors have been described, all targeting the mecA gene: a nanoelectrokinetic sensor [ 138 ], a capacitive biosensor tested on a saliva sample [ 139 ], a portable and smartphone-controlled colorimetric LAMP device [ 140 ], a hairpin probe-mediated DNA circuit based on exonuclease III and DNAzyme tested on mecA -spiked serum samples [ 141 ], a colorimetric paper-based analytical device [ 142 ], a ligation chain reaction electrochemical sensor tested on joint fluid samples [ 143 ] and a microfluidic biochip based on roll-to-roll UV nanoimprint technology tested on nasal, throat and inguinal samples [ 144 ].…”

Advances in the Application of Nanomaterials as Treatments for Bacterial Infectious Diseases

“…An electrochemiluminescent lateral flow immunosensor (ECL-LFI), Ru(bpy)32+-labeled AuNPs, has been used to detect CRP [67]. Another multiplex loop-mediated isothermal amplification coupled with a NP-based lateral flow biosensor (m-LAMP-LFB) has been used in detection of all S. aureus species [68]. The LAMP-LFB also provides a reliable and rapid assay for E. faecalis, Neisseria meningitidis and M. pneumoniae detection, which is significant for diagnosis and follow-up treatment [62,69,70].…”

“…As mentioned above the m-LAMP-LFB was applied not only to detect all S. aureus strains, but also to detect methicillin-resistant genes from all S. aureus species [68]. Multiplex MCDA (m-MCDA), which aims to detect the nuc gene (S. aureus-specific gene) and mecA gene (encoding penicillin-binding protein-2 ), has been shown to detect S. aureus strains and MRSA within 85 min [68]. Fast dissemination of the colistin resistance (mcr) gene mcr-1 in Enterobacteriaceae is very important in combating bacterial infections [80].…”